Adiponectin receptor and gene encoding the same

ABSTRACT

The object is to isolate and identify human and mouse adiponectin receptors, to provide a novel protein having adiponectin binding ability, and to provide a screening method and screening kit for a ligand, agonist and antagonist to an adiponectin receptor using such protein. To achieve this object, a protein is used, as novel protein having adiponectin binding ability, that is (a) a protein comprising an amino acid sequence according to Seq. No. 2, 4, 6 or 8, or (b) a protein comprising an amino acid sequence according to Seq. No. 2, 4, 6 or 8 with one or more amino acids deleted, replaced or added, and having adiponectin binding ability.

RELATED APPLICATIONS

[0001] This application is a continuation of PCT/JP03/07515 entitled“Adiponectin Receptor and Gene Encoding the Same”, filed Jun. 12, 2003,which claims priority to Japanese Application No. 2002-383738 filed Dec.29, 2002. Each of the aforementioned applications are herebyincorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] The present invention relates to a novel protein havingadiponectin-binding ability, a gene encoding the protein, a recombinantvector containing the gene, a transformant containing the recombinantvector, and an antibody to the protein and a fragment of the antibody.Moreover, the present invention relates to a screening method and ascreening kit for a ligand, agonist or antagonist to an adiponectinreceptor.

BACKGROUND ART

[0003] Obesity is defined as an increased mass of adipose tissue, and isassociated with a higher risk of cardiovascular and metabolic disorderssuch as diabetes, hyperlipidemia and coronary heart disease (Reaven, G.M., Diabetologia 38, 3-13 (1995); Spiegelman, B. M. et al, Cell 87,377-389 (1996)). Impaired glucose and lipid metabolism, a hallmark ofobesity and type 2 diabetes, causes increased lipid storage in insulintarget tissues such as muscle and liver, thereby leading to insulinresistance (Ruderman, N. B. et al, Am. J. Physiol. 276, E1-E18 (1999);Shulman, G. I., J. Clin. Invest. 106, 171-176 (2000)). The adiposetissue itself serves as the site of triglyceride (TG) storage and freefatty acids (FFA)/glycerol release in response to changing energydemands (Spiegelman, B. M. and Flier, J. S., Cell 87, 377-389 (1996)).Adipose tissue also participates in the regulation of various types ofenergy homeostasis as an important endocrine organ that secretes anumber of biologically active substances called “adipokines” (Matsuzawa,Y. et al, Ann. NY Acad. Sci. 892, 146-154 (1999)) such as FFA (Shulman,G. I., J. Clin. Invest. 106, 171-176 (2000)), adipsin (White, R. T. etal, J. Biol. Chem. 267, 9210-9213 (1992)), leptin (Friedman, J. M.,Nature 404, 632-634 (2000)), plasminogen activator inhibitor-1 (PAI-1)(Shimomura, I. et al, Nat. Med. 2, 800-803 (1996)), resistin (Steppan,C. M. et al, Nature 409, 307-312 (2001)) and tumor necrosis factor-α(TNF-α) (Hotamisligil, G. S., J. Intern. Med. 245, 621-625 (1999)).

[0004] Adiponectin or Acrp30 (Hu, E., Liang, P. et al, J. Biol. Chem.271, 10697-10703 (1996) and others) is an adipocyte-derived hormone withmultiple biological functions. It has been reported that obesity, type 2diabetes and coronary heart disease are associated with decreased plasmaadiponectin levels, and that adiponectin may have putativeanti-atherogenic properties in vitro (Ouchi, N. et al, Circulation 103,1057-1063 (2001); Yokota, T. et al, Blood 96, 1723-1732 (2000)). Also,it has been reported that an acute increase in circulating levels ofAcrp30 lowers hepatic glucose production (Berg, A. H. et al, Nat. Med.7, 947-953 (2001); Combs, T. P. et al, J. Clin. Invest. 108, 1875-1881(2001)). Also, it has been reported that globular Acrp30 increases fattyacid oxidation in muscle, and causes weight loss in mice (Fruebis, J. etal, Proc. Natl. Acad. Sci. USA 98, 2005-2010 (2001)). Also, it has beenreported that treatment with adiponectin consisting solely of theglobular domain (globular adiponectin or gAd) increases fatty acidoxidation in muscle, thereby ameliorating insulin resistance inlipoatrophic mice and obese mice, while treatment with full-lengthadiponectin also ameliorates though less than with gAd (Yamauchi, T. etal, Nat. Med. 7, 941-946 (2001)).

[0005] Recently it has been reported that adiponectin acutely activatesAMP kinase (AMPK) in skeletal muscle, thus stimulating fatty acidoxidation and glucose uptake (Yamauchi, T. et al, Nat. Med. 8, 1288-1295(2002)), and that adiponectin chronically activates PPARα, resulting inincreased fatty acid oxidation but reduced tissue TG content in themuscles, with these effects being greater with gAd than with full-lengthadiponectin (Yamauchi, T. et al, J. Biol. Chem. 278, 2461-2468 (2002)).Interestingly, in the liver full-length adiponectin alone acutelyactivates AMPK, causing a reduction in gluconeogenesis-associatedmolecules and stimulating fatty-acid oxidation, and moreover full-lengthadiponectin alone chronically activates AMPK, stimulating fatty-acidoxidation and reducing tissue TG levels in the liver. All these changesserve to enhance insulin sensitivity in vivo (Yamauchi, T. et al, Nat.Med. 8, 1288-1295 (2002); Yamauchi, T. et al, J. Biol. Chem. 278,2461-2468 (2002)).

[0006] These effects of adiponectin are believed to be mediated byreceptors on the cell surface, but adiponectin receptors have not beenidentified, and it is unknown whether the adiponectin receptors in theskeletal muscle and liver differ either structurally or functionally.The inventors identified a gene encoding adiponectin receptors, anddiscovered from a homology search that yeast YOL002c gene is a homologue(Karpichev, I. V. et al, Journal of Biological Chemistry 277,19609-19617 (2002)). YOL002c encodes a seven transmembrane protein thatplays a key role in the metabolic pathways of lipids, such as fatty acidoxidation (Karpichev, I. V. et al, Journal of Biological Chemistry 277,19609-19617 (2002)).

DISCLOSURE OF THE INVENTION

[0007] First, it is an object of the present invention to provide anovel protein having adiponectin-binding ability, a gene encoding theprotein, a recombinant vector containing the gene, a transformantcontaining the recombinant vector, and an antibody to the protein and afragment of the antibody.

[0008] Second, it is an object of the present invention to provide ascreening method and screening kit for a ligand, agonist or antagonistto an adiponectin receptor.

[0009] In order to achieve these objects, the present invention providesthe following protein, gene, recombinant vector, transformant andantibody, as well as a screening method and screening kit for a ligand,agonist or antagonist to an adiponectin receptor.

[0010] (1) A protein as set forth in (a) or (b) below:

[0011] (a) a protein comprising an amino acid sequence according to Seq.No. 2, 4, 6 or 8; or

[0012] (b) a protein comprising an amino acid sequence according to Seq.No. 2, 4, 6 or 8 with one or more amino acids deleted, replaced oradded, and having adiponectin binding ability.

[0013] (2) A gene encoding the protein according to the abovementioned(1).

[0014] (3) The gene according to the abovementioned (2), comprising DNAas set forth in (c) or (d) below:

[0015] (c) DNA comprising a base sequence according to Seq. No. 1, 3, 5or 7; or

[0016] (d) DNA which hybridizes under stringent conditions with DNAcomplementary to DNA comprising a base sequences according to Seq. No.1, 3, 5 or 7, and which encodes a protein having adiponectin bindingability.

[0017] (4) A recombinant vector containing the gene according to theabovementioned (2) or (3).

[0018] (5) A transformant containing the recombinant vector according tothe abovementioned (4).

[0019] (6) An antibody or a fragment thereof capable of reacting withthe protein according to the abovementioned (1).

[0020] (7) A screening method for a ligand, agonist or antagonist to anadiponectin receptor, comprising a step of bringing a test substanceinto contact with the protein according to the abovementioned (1).

[0021] (8) A screening kit for a ligand, agonist or antagonist to anadiponectin receptor, comprising the protein according to theabovementioned (1), the DNA according to the abovementioned (2) or (3),and the recombinant vector according to abovementioned (4) or thetransformant according to abovementioned (5).

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1(a) shows the binding of adiponectin to C2C12 myocytes, FIG.1(b) shows the binding of adiponectin to hepatocytes, FIGS. 1(c) through1(e) show the results of FACS analysis, FIG. 1(f) shows a schematic viewof the structure of AdipoR1 and AdipoR2 genes transcripts, FIG. 1(g)shows the results of northern blot analysis for various mouse tissues,and FIG. 1(h) shows the results of northern blot analysis for varioushuman tissues;

[0023]FIG. 2(a) shows a schematic view of the structures of AdipoR1 andAdipoR2, FIG. 2(b) shows the results of immunoblotting with anti-FLAGantibodies of cell lysates from 293T cells transfected with AdopR1 andAdipoR2 having the epitope tag FLAG, FIG. 2(c) shows the intracellularlocations of AdipoR1 or AdipoR2 in 293T cells transfected with AdipoR1and AdipoR2 having epitope flags introduced at the N or C end, and FIGS.2(d) and 2(e) show the predicted structural models of Adipo R1 andAdipoR2, respectively;

[0024]FIG. 3(a) shows the binding isotherm of [¹²⁵I] globular Adipo(gAd) binding to 293T cells transfected with AdipoR1 or AdipoR2, FIG.3(b) shows the binding isotherm of [¹²⁵I] full-length Adipo (Ad) bindingto the 293T cells, FIG. 3(c) shows the results when cell lysates of 293Tcells transfected with AdipoR1 or AdipoR2 having the epitope tag FLAG orHA are immunoprecipitated with an anti-FLAG or anti-HA antibody, andthen immunoblotted with the anti-FLAG or anti-HA antibody, FIG. 3(d)shows changes in [Ca²⁺]i when mouse AdipoR1-expressing cells orBLT1-expressing cells were challenged with globular Adipo, full-lengthAdipo, LTB4 or ATP, FIG. 3(e) shows the results for accumulation of cAMPor cGMP in HEK-29 cells treated with or without forskolin, and FIG. 3(f)shows PPARα ligand activity in 293T cells transfected with AdipoR1 thatare incubated with globular Adipo or full-length Adipo;

[0025]FIG. 4(a) shows mouse AdipoR1 mRNA levels, FIG. 4(b) shows mouseAdipoR2 mRNA levels, FIGS. 4(c) and 4(d) show binding isotherms of[¹²⁵I] globular Adipo (gAd) or full-length Adipo (Ad) binding to C2C12myocytes transfected with mouse AdipoR1 or AdipoR2, FIG. 4(e) showsPPARα ligand activity in C2C12 myocytes transfected with mouse AdipoR1or Adipo R2 which were infected with an adenovirus containing LacZ orDN-α2AMPK and treated for 7 hours with globular Adipo or full-lengthAdipo, and FIG. 4(f) shows in vitro fatty acid oxidation in the C2C12myocytes;

[0026]FIG. 5(a) shows mouse AdipoR1 mRNA levels in C2C12 myocytestransfected with siRNA or mock, FIG. 5(b) shows mouse AdipoR2 mRNAlevels in the C2C12 myocytes, FIG. 5(c) shows the results of acompetitive radioligand binding assay in which [¹²⁵I] globular Adipobinding to cells transfected with siRNA dublex is displaced byincreasing concentrations of unlabeled globular Adipo, FIG. 5(d) showsthe results of a competitive radioligand binding assay in which [¹²⁵I]full-length Adipo binding to cells transfected with siRNA duplex isdisplaced by increasing concentrations of unlabeled full-length Adipo,FIG. 5(e) shows the binding isotherm of [¹²⁵I] globular Adipo binding toC2C12 myocytes transfected with siRNA duplex, FIG. 5(f) shows thebinding isotherm of [¹²⁵I] full-length Adipo binding to C2C12 myocytestransfected with siRNA duplex, FIG. 5(g) shows PPARα ligand activity inC2C12 myocytes transfected with siRNA duplex which are incubated for 7hours with globular Adipo, full-length Adipo or Wy-14,643, FIG. 5(h)shows in vitro fatty acid oxidation in C2C12 myocytes transfected withsiRNA duplex which are incubated for 7 hours with globular Adipo,full-length Adipo or Wy-14,643, and FIG. 5(i) shows glucose uptake inC2C12 myocytes transfected with siRNA duplex which are incubated for 7hours with globular Adipo, full-length Adipo or insulin;

[0027]FIG. 6(a) shows specific binding of gAd to hepatocytes, FIG. 6(b)shows specific binding of Ad to hepatocytes, FIG. 6(c) shows expressionlevels of human AdipoR1 mRNA in HAEC, FIG. 6(d) shows expression levelsof human AdipoR2 mRNA in HAEC, FIG. 6(e) shows specific binding of gAdto HAEC, and FIG. 6(f) shows specific binding of Ad to HAEC;

[0028]FIG. 7(a) shows the binding isotherm of [¹²⁵I] globular Adipobinding to C2C12 cells transfected with siRNA duplex, FIG. 7(b) showsthe binding isotherm of [¹²⁵I] full-length Adipo binding to C2C12myocytes transfected with siRNA duplex, FIG. 7(c) shows the results ofScatchard plot analysis based on the results shown in FIG. 7(a), andFIG. 7(d) shows the results of Scatchard plot analysis based on theresults shown in FIG. 7(b); and

[0029]FIG. 8(a) shows the results when C2C12 cells transfected or nottransfected with AdipoR1 are incubated for 10 minutes with 0.1 μg/mL or1 μg/mL of gAd and a lysate of the cells was then reacted withanti-phosphorylated AMPK antibodies, FIG. 8(b) shows the results whenC2C12 cells transfected or not transfected with AdipoR1 are incubatedfor 10 minutes with 0.1 μg/mL or 1 μg/mL of gAd and a lysate of thecells is then reacted with anti-phosphorylated ACC antibodies, FIG. 8(c)shows the results when C2C12 cells transfected or not transfected withAdipoR1 are incubated for 10 minutes with 0.1 μg/mL or 1 μg/mL of gAdand a lysate of the cells is then reacted with anti-phosphorylated p38MAPK antibodies, FIG. 8(d) shows the results when C2C12 cellstransfected or not transfected with AdipoR1 are incubated for 10 minuteswith 0.1 μg/mL or 1 μg/mL of gAd and a lysate of the cells is thenreacted with anti-phosphorylated MAPK antibodies, FIG. 8(e) shows theresults when hepatocytes transfected or not transfected with AdipoR1were incubated for 10 minutes with 0.1 μg/mL or 1 μg/mL of gAd and alysate of the cells was then reacted with anti-phosphorylated AMPKantibodies, FIG. 8(f) shows the results when hepatocytes transfected ornot transfected with AdipoR1 are incubated for 10 minutes with 0.1 μg/mLor 1 μg/mL of gAd and a lysate of the cells is then reacted withanti-phosphorylated ACC antibodies, FIG. 8(g) shows fatty acid oxidationof C2C12 cells transfected or not transfected with AdipoR1 in thepresence of dominant negative AMP kinase (DN-AMPK) or p38 MAPK-specificinhibitor SB203580, and FIG. 8(h) shows glucose intake of C2C12 cellstransfected or not transfected with AdipoR1 in the presence of dominantnegative AMP kinase (DN-AMPK) or p38 MAPK-specific inhibitor SB203580.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] A protein of the present invention is a protein as set forth in(a) or (b) below:

[0031] (a) a protein comprising an amino acid sequences according toSeq. No. 2, 4, 6 or 8 (hereunder “protein

[0032] (a)”);

[0033] (b) a protein comprising an amino acid sequences according toSeq. No. 2, 4, 6 or 8 with one or more amino acids deleted, replaced oradded, and having adiponectin binding ability (hereunder “protein (b)”).

[0034] Protein (a) or (b) has adiponectin binding ability. “Adiponectinbinding ability” signifies the ability to bind with adiponectin, andpreferably the ability to bind specifically with adiponectin (that is,the adiponectin binding ability of an adiponectin receptor).“Adiponectin binding ability” includes binding ability with respect toeither full-length adiponectin or adiponectin consisting only of theglobular domain. Proteins having adiponectin binding ability includeproteins that can bind only to full-length adiponectin, proteins thatcan bind only to adiponectin consisting only of the globular domain,proteins that can bind to both full-length adiponectin and adiponectinconsisting only of the globular domain, and proteins which bindpreferentially to either full-length adiponectin or adiponectinconsisting only of the globular domain.

[0035] The “globular domain” is the filament-like structural domain atthe C end of adiponectin, having a length of a little over 100 aminoacids. Complement C1q and the like have domains highly homologous to theglobular domain. Functionally, the globular domain of adiponectin actsstrongly on skeletal muscle and the like, combusting fatty acids andinhibiting the storage of lipids, and it exhibits the same level ofactivity as full-length adiponectin at a concentration as low asone-several tenth of that of the full-length adiponectin.

[0036] Of proteins (a), those according to Seq. No. 2 or 4 are humanadiponectin receptors, while those according to Seq. No. 6 or 8 aremouse adiponectin receptors. The proteins according to Seq. Nos. 2 and 6are the same kind of adiponectin receptor (AdipoR1), while thoseaccording to Seq. Nos. 4 and 8 are a kind of adiponectin receptor(AdipoR2) different from AdipoR1. AdipoR1 is thought to be a receptorwith relative selectivity for adiponectin consisting solely of theglobular domain rather than full-length adiponectin, while AdipoR2 isthought to be a receptor with relative selectivity for full-lengthadiponectin rather than adiponectin consisting solely of the globulardomain.

[0037] The degree of homology between human (Seq. No. 2) and mouse (Seq.No. 6) AdipoR1 at the amino acid level is 96.8%. The degree of homologybetween human (Seq. No. 4) and mouse (Seq. No. 8) AdipoR2 at the aminoacid level is 95.2%. The structures of AdipoR1 and AdipoR2 are similar,and the homology between AdipoR1 (Seq. No. 6) and AdipoR2 (Seq. No. 8)in mice is 66.7%.

[0038] In vivo, AdipoR1 is expressed in most tissues, with especiallyhigh expression in skeletal muscle, while AdipoR2 is expressed stronglyin the liver. AdipoR1 and AdipoR2 are thought to form both homo- andhetero-multimers. Both AdipoR1 and AdipoR2 bind with both full-lengthadiponectin and adiponectin consisting solely of the globular domain,mediating the stimulating effects of these adiponectins on PPARα(Peroxisome proliferator-activated receptor a) ligand activity and fattyacid oxidation. For example, AdipoR1 is thought to mediate the increasesin PPARα ligand activity, fatty acid oxidation and glucose uptake byadiponectin consisting only of the globular domain in myocytes.Similarly, AdipoR2 is thought to partially mediate the increases inPPARα ligand activity and fatty acid oxidation by full-lengthadiponectin in hepatocytes and myocytes.

[0039] AdipoR1 and AdipoR2 Are both thought to contain seventransmembrane domains. In the amino acid sequence according to Seq. No.2 (human AdipoR1), the amino acid sequences 136-158, 172-194, 207-228,234-255, 267-289, 299-321 and 336-358 are though to correspond totransmembrane domains. In the amino acid sequence according to Seq. No.4 (human AdipoR2), the amino acids sequences 60-82, 96-118, 130-152,158-179, 192-214, 222-244 and 260-282 are though to correspond totransmembrane domains. In the amino acid sequence according to Seq. No.6 (mouse AdipoR1), the amino acid sequences 136-158, 172-194, 207-228,234-255, 267-289, 299-321 and 336-358 are though to correspond totransmembrane domains. Finally, in the amino acid sequence according toSeq. No. 8 (mouse AdipoR2), the amino acid sequences 72-94, 108-130,142-164, 170-191, 204-226, 234-256 and 272-294 are though to correspondto transmembrane domains.

[0040] There are no particular limits on the number of amino acidsdeleted, replaced or added in the amino acid sequences according to Seq.Nos. 2, 4, 6 and 8 as long as adiponectin binding ability is retained.The number may be one or more than one or preferably one or a few, orspecifically in the range of normally 1-100 or preferably 1-50 or morepreferably 1-10. The amino acid sequences of protein (b) should normallyhave 60% or more, or preferably 80% or more, or more preferably 90% ormore homology with the amino acid sequence of protein (a).

[0041] There are no particular limits on the locations of the aminoacids deleted, replaced or added in the amino acid sequences accordingto Seq. No. 2, 4, 6 or 8, as long as adiponectin binding ability isretained.

[0042] Protein (b) includes proteins wherein deletions, replacements,additions and other mutations have been artificially introduced into aprotein (a), as well as proteins naturally occurring with introduceddeletions, replacements, additions and other mutations and such proteinswith deletions, replacements, additions and other mutations artificiallyintroduced. Examples of proteins naturally occurring with introduceddeletions, replacements, additions and other mutations include proteinsderived from humans and other mammals such as monkeys, cows, sheep,goats, horses, pigs, rabbits, dogs, cats, mice, rats and the like(including proteins which may occur due to polymorphisms in suchmammals).

[0043] Proteins (a) and (b) include both proteins with added sugarchains and proteins without added sugar chains. The types, locations andthe like of the sugar chains added to the proteins will differ dependingon the type of host cells used in manufacturing the protein, butproteins with added sugar chains include proteins obtained using anykind of host cells. Proteins (a) and (b) also include pharmacologicallyallowable salts thereof.

[0044] A gene encoding protein (a) or (b) can be obtained for example byusing mRNA extracted from the skeletal muscle, livers, hearts,macrophages, blood vessels, brains, kidneys, lungs, placentas, spleens,testes, peripheral blood, thymus glands, intestines or other tissues ofhumans, mice or other mammals to build a cDNA library, and then usingprobes synthesized based on the base sequences according to Seq. No. 1,3, 5 or 7 to screen the cDNA library for clones containing the targetDNA. The steps involved in preparing a cDNA library and screening forclones containing the target DNA are explained below.

[0045] [Preparing a cDNA Library]

[0046] To prepare a cDNA library, total RNA is first obtained from theskeletal muscle, livers, hearts, macrophages, blood vessels, brains,kidneys, lungs, placentas, spleens, testes, peripheral blood, thymusglands, intestines or other tissues of humans, mice or other mammals forexample, and poly(A+)RNA (mRNA) is then obtained by the batch method,affinity column method or the like using oligo(dT)-cellulose,poly(U)-sepharose or the like. The poly(A+)RNA (mRNA) in this case mayalso be fractionated by sucrose density gradient centrifugation or thelike. Taking the resulting mRNA as the template, single-stranded cDNA isthen synthesized using oligo(dT) primer and reverse transcriptase, anddouble-stranded cDNA is synthesized from the single-stranded cDNA. Theresulting double-stranded cDNA is incorporated into a suitable cloningvector to prepare a recombinant vector which is used to transform E.coli or other host cells, and a cDNA library is obtained by selection oftransformants using tetracycline resistance or ampicillin resistance asthe indicator. The cloning vector for preparing the cDNA library may beany vector that can replicate independently in the host cells, and forexample phage vectors, plasmid vectors and the like can be used. E. colior the like can be used as the host cells.

[0047] Transformation of the E. coli or other host cells can beaccomplished by a method such as adding the recombinant vector tocompetent cells prepared with calcium chloride, magnesium chloride orrubidium chloride. When a plasmid is used as the vector, a resistancegene for a drug such as tetracycline or ampicillin is included.

[0048] A commercial kit such as the SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning (Gibco BRL) or the ZAP-cDNA Synthesis Kit(Stratagene) can be used in preparing the cDNA library.

[0049] [Screening for Clones Containing the Target DNA]

[0050] To screen the cDNA library for clones containing the target DNA,primer is synthesized based on the base sequence according to Seq. No.1, 3, 5 or 7, and used in a polymerase chain reaction (PCR) to obtainPCR amplified fragments. The PCR amplified fragments may also besub-cloned using a suitable plasmid vector. There are no particularlimits on the primer set used in PCR, which can be designed based on thebase sequence according to Seq. Nos. 1, 3, 5 or 7.

[0051] The target DNA is obtained by colony hybridization or plaquehybridization of the cDNA library, with the PCR amplified fragments asthe probe. The PCR amplified fragments may be labeled with an isotope(such as ³²P or ³⁵S), biotin, digoxigenin, alkaliphosphotase or the likefor use as the probe. Clones containing the target DNA are obtained byan expression screening method such as immunoscreening using antibodies.

[0052] The resulting DNA can be incorporated into a vector by ordinarymethods, either as the original DNA fragments or after cleavage with asuitable restriction enzyme or the like, and the base sequence can bedetermined by an ordinarily used method of base sequence analysis, suchas the Maxam-Gilbert chemical modification method or thedideoxynucleotide chain termination method. A base sequence analyzingdevice such as the 373A DNA Sequencer (Perkin Elmer) is normally used inanalyzing the base sequence.

[0053] A gene encoding protein (a) or (b) comprises an open readingframe encoding protein (a) or (b) and the termination codon located atthe 3′ end thereof. Moreover, a gene encoding protein (a) or (b) cancomprise an untranslated region (UTR) at the 5′ and/or 3′ ends of theopen reading frame.

[0054] Examples of the gene encoding protein (a) include a genecomprising DNA comprising a base sequence according to Seq. No. 1, 3, 5or 7. Of the base sequence according to Seq. No. 1, bases 1-1125constitute the open reading frame which encodes the protein according toSeq. No. 2, while the translation initiation codon is located at bases1-3 of the base sequence according to Seq. No. 1, and the terminationcodon at bases 1126-1128 of the sequence. Of the base sequence accordingto Seq. No. 3, bases 1-897 constitute the open reading frame whichencodes the protein according to Seq. No. 4, while the translationinitiation codon is located at bases 1-3 of the base sequence accordingto Seq. No. 3, and the termination codon at bases 898-900. Of the basesequence according to Seq. No. 5, bases 1-1125 constitute the openreading frame which encodes the protein according to Seq. No. 6, whilethe translation initiation codon is located at bases 1-3 of the basesequence according to Seq. No. 5, and the termination codon at bases1126-1128. Of the base sequence according to Seq. No. 7, bases 1-933constitute the open reading frame which encodes the protein according toSeq. No. 8, while the translation initiation codon is located at bases1-3 of the base sequence according to Seq. No. 7, and the terminationcodon at bases 934-936.

[0055] There are no particular limits on the base sequence of the geneencoding protein (a) so long as it encodes protein (a), and the basesequence of the open reading frame is not restricted to a base sequenceaccording to Seq. No. 1, 3, 5 or 7. The gene encoding protein (a) canalso be obtained by chemical synthesis according to its base sequence.Chemical synthesis of DNA can be accomplished using a commercial DNAsynthesizer, such as a DNA synthesizer using the thiophosphite method(Shimadzu Corporation) or the phosphoramidite method (Perkin Elmer).

[0056] An example of a gene encoding protein (b) is a gene comprisingDNA which hybridizes under stringent conditions with DNA complementaryto DNA comprising a base sequence according to Seq. No. 1, 3, 5 or 7,and which encodes a protein having adiponectin binding ability.

[0057] An example of “stringent conditions” would be for example 42° C.,2×SSC and 0.1% SDS, or preferably 65° C., 0.1×SSC and 0.1% SDS.

[0058] A specific example of DNA which hybridizes under stringentconditions with DNA complementary to DNA comprising a base sequenceaccording to Seq. No. 1, 3, 5 or 7 is DNA having 60% or more orpreferably 80% or more or more preferably 90% or more homology with abase sequence according to Seq. No. 1, 3, 5 or 7.

[0059] The gne encoding protein (b) can be obtained for example byartificially introducing mutations into DNA comprising a base sequenceaccording to Seq. No. 1, 3, 5 or 7, using a well-known method such assite-directed mutagenesis. Mutations can be introduced using amutagenesis kit, such as a Mutant-K (Takara), Mutant-G (Takara) orTakara LA PCR in vitro Mutagenesis series kit. DNA whose base sequencehas already been determined can be obtained by chemical synthesis.

[0060] Protein (a) or (b) can be manufactured according to the followingsteps by expressing the gene encoding each protein in host cells.

[0061] [Preparation of Recombinant Vectors and Transformants]

[0062] To prepare a recombinant vector, a DNA fragment of a suitablelength is prepared which comprises a region encoding the target protein.The DNA is also prepared wherein bases of the base sequence of theprotein-encoding region are replaced so that the codons are optimizedfor expression in the host cells.

[0063] A recombinant vector is prepared by inserting this DNA fragmentdownstream from the promoter of a suitable expression vector, and atransformant capable of producing the target protein is obtained byintroducing the recombinant vector into suitable host cells. The DNAfragment needs to be incorporated into the vector so that its functionsare expressed, and the vector may contain in addition to the promoterenhancers or other cis-elements, splicing signals, poly(A) additionsignals, selection markers (such as dihydrofolate reductase gene,ampicillin resistance gene or neomycin resistance gene), ribosomebinding sequences (SD sequences) or the like.

[0064] There are no particular limits on the expression vector as longas it can replicate independently in the host cells, and for exampleplasmid vectors, phage vectors, virus vectors and the like can be used.Examples of plasmid vectors include E. coli-derived plasmids (such aspRSET, pBR322, pBR325, pUC118, pUC119, pUC18 and pUC19), Bacillussubtilis-derived plasmids (such as pUB110 and pTP5) and yeast-derivedplasmids (such as YEp13, YEp24 and Ycp50). Examples of phage vectorsinclude λ-phages (such as Charon4A, Charon21A, EMBL3, EMBL4, λgt10,λgt11 and λZAP), and examples of virus vectors include retroviruses,vaccinia virus and other animal viruses and baculoviruses and otherinsect viruses.

[0065] As long as the host cells can express the target DNA, they can beprokaryotic cells, yeasts, animal cells, insects cells, plant cells orthe like. An animal body, plant body, silkworm body or the like can alsobe used.

[0066] When bacteria are used as the host cells, they can be for exampleEscherichia coli or other Escherichia, Bacillus subtilis or otherBacillus, Pseudomonas putida or other Pseudomonas, or Rhizobium melilotior other Rhizobium bacteria. Specifically, Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli K12,Escherichia coli JM109, Escherichia coli HB101 and other Escherichiacoli and Bacillus subtilis MI 114, Bacillus subtilis 207-21 and otherBacillus subtilis can be used as the host cells. There are no particularlimits on the promoter in this case as long as it is capable ofexpression in E. coli or other bacteria, and for example a trp promoter,lac promoter, P_(L) promoter, P_(R) promoter or other E. coli-orphage-derived promoter can be used. Artificially designed and alteredpromoters such as tac promoter, lacT7 promoter or let I promoter mayalso be used.

[0067] There are no particular limits on the method of introducing therecombinant vector into the bacteria as long as the method can introduceDNA into bacteria, and electroporation or a method employing calciumions may be used for example.

[0068] When a yeast is used for the host cells, Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris or the like maybe used. There are no particular limits on the promoter in this case aslong as it is capable of expression in yeasts, and for example a gal1promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter,PH05 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoteror the like can be used.

[0069] There are no particular limits on the method of introducing therecombinant vector into the yeast as long as the method can introduceDNA into yeast, and the electroporation, spheroplast or lithium acetatemethod or the like may be used.

[0070] When the host cells are animals cells, monkey COS-7 cells, Vero,chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, humanFL cells or the like may be used as the host cells. There are noparticular limits on the promoter in this case as long as it is capableof expression in animal cells, and for example an SRα promoter, SV40promoter, LTR (Long Terminal Repeat) promoter, CMV promoter or humancytomegalovirus early gene promoter or the like can be used.

[0071] There are no particular limits on the method of introducing therecombinant vector into the animal cells as long as the method canintroduce DNA into animal cells, and the electroporation, calciumphosphate or lipofection method or the like can be used.

[0072] When the host cells are insect cells, Spodoptera frugiperdaovarian cells, Trichoplusia ni ovarian cells, cultured cells fromsilkworm ovaries and the like may be used as the host cells. PossibleSpodoptera frugiperda ovarian cells include Sf9 and Sf21 cells and thelike, Trichoplusia ni ovarian cells include High 5 and BTI-TN-5B1-4(Invitrogen) and the like, and cultured cells derived from silkwormovaries include Bombyx mori N4 cells and the like.

[0073] There are no particular limits on the method of introducing therecombinant vector into the insect cells as long as the method canintroduce DNA into insect cells, and the calcium phosphate, lipofectionor electroporation method or the like can be used.

[0074] [Culturing the Transformant]

[0075] A transformant into which a recombinant vector with incorporatedDNA encoding the target protein has been introduced can be cultured byordinary culture methods. Culturing of the transformant can beaccomplished by ordinary methods used in the culture of host cells.

[0076] The medium for culturing a transformant obtained as bacteria,yeasts or other microbial host cells contains carbon sources, nitrogensources, inorganic salts and the like which can be used by thosemicroorganisms, and either a natural or synthetic medium may be used aslong as it is a medium for efficient culturing of the transformant.

[0077] Glucose, fructose, sucrose, starch and other carbohydrates,acetic acid, propionic acid and other organic acids, and ethanol,propanol and other alcohols can be used as carbon sources. Ammonia,ammonium chloride, ammonium sulfate, ammonium acetate, ammoniumphosphate and other ammonium salts of inorganic and organic acids aswell as peptone, meat extract, yeast extract, corn steep liquor, caseinhydrolysate and the like can be used as nitrogen sources. Monopotassiumphosphate, dipotassium phosphate, magnesium phosphate, magnesiumsulfate, sodium chloride, ferrous sulfate, manganese sulfate, coppersulfate, calcium carbonate and the like can be used as inorganic salts.

[0078] Culturing of a transformant obtained as E. coli, yeast or othermicrobial host cells is accomplished under aerobic conditions such as ashaking culture or aerated spinner culture. The culture temperature isnormally 25-37° C., the culture time is normally 12-48 hours, and a pHof 6-8 is maintained during the culture period. The pH can be adjustedusing an inorganic acid, organic acid or alkaline solution or urea,calcium carbonate, ammonia or the like. An antibiotic such as ampicillinor tetracycline can be added to the medium as necessary during culture.

[0079] When culturing microorganisms transformed with an expressionvector employing an inducible promoter as the promoter, an inducer canbe added to the medium as necessary. For example,isopropyl-β-D-thiogalactopyranoside or the like can be added to themedium when culturing a microorganism transformed with an expressionvector employing a lac promoter, while indoleacrylic acid or the likecan be added when culturing a microorganism transformed with anexpression vector employing a trp promoter.

[0080] Commonly used RPMI1640 medium, Eagle MEM medium, DMEM medium, HamF12 medium, Ham F12K medium and such medium with fetal calf serum addedcan be used as the medium for culturing a transformant obtained withanimal cells as the host cells. The transformant is normally culturedfor 3-10 days at 37° C. under the presence of 5% CO₂. For purposes ofculture, an antibiotic such as kanamycin, penicillin, streptomycin orthe like can be added to the medium as necessary.

[0081] Commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium(Gibco BRL), ExCell400 or ExCell405 (JRH Biosciences) or the like can beused as the medium for culturing a transformant obtained with insectcells as the host cells. The transformant is normally cultured for 3-10days at 27° C. For purposes of culture, an antibiotic such as gentamicincan be added to the medium as necessary.

[0082] The target protein can also be expressed as an excreted proteinor a fused protein. Examples of proteins to be fused includeβ-galactosidase, protein A, protein A IgG binding region,chloramphenicol acetyltransferase, poly(Arg), poly(Glu), protein G,maltose binding protein, glutathione S transferase, polyhistidine chain(His-tag), S peptide, DNA binding protein domain, Tac antigen,thioredoxin, and green fluorescent protein.

[0083] [Protein Isolation and Purification]

[0084] The target protein is obtained by harvesting the target proteinfrom the transformant culture. “Culture” in this case includes culturesupernatant, cultured cells, cultured bacterial bodies, or crushed cellsor bacterial cell bodies.

[0085] When the target protein is accumulated in the transformant cells,the cells in the culture are collected by centrifugation of the culture,and the cells are then washed and crushed and the target proteinextracted. If the target protein is excreted outside the transformantcells, the culture supernatant is used as is, or else cells or bacterialcell bodies are removed from the culture supernatant by centrifugationor the like.

[0086] The resulting protein (a) or (b) can then be purified by a methodsuch as solvent extraction, salting-out desalting with ammonium sulfateor the like, precipitation with organic solvents, diethylaminoethyl(DEAE)-sepharose, ion exchange chromatography, hydrophobicchromatography, gel filtration, affinity chromatography or the like.

[0087] Protein (a) or (b) can also be manufactured based on its aminoacid sequence by a chemical synthesis method such as the Fmoc(fluorenylmethyloxycarbonyl) or tBoc (t-butyloxycarbonyl) method. Acommercial peptide synthesizer can be used in this case.

[0088] The antibodies or fragments thereof of the present invention areantibodies or fragments thereof which react to protein (a) or (b). Asused here, “antibodies” include both monoclonal and polyclonalantibodies, and “monoclonal and polyclonal antibodies” include allclasses of monoclonal and polyclonal antibodies. “Antibodies” alsoinclude antiserum obtained by immunizing rabbits, mice or other immuneanimals with protein (a) or (b), as well as human antibodies andhumanized antibodies obtained by genetic recombination. “Antibodyfragments” include Fab fragments, F(ab)′₂ fragments, single-chainantibodies (scFv) and the like.

[0089] The antibodies or fragments thereof of the present invention areprepared using protein (a) or (b) as the immunizing antigen. Forexample, (i) crushed or crushed and purified cells or tissue expressingprotein (a) or (b), (ii) recombinant protein expressed by introductionof DNA encoding protein (a) or (b) into E. coli, insect cells, animalcells or another host by genetic recombination, or (iii) chemicallysynthesized peptides or the like can be used as the immunizing antigen.

[0090] To prepare polyclonal antibodies, rats, mice, guinea pigs,rabbits, sheep, horses, cows or other mammals are immunized with theimmunizing antigen. It is preferable to use mice as the immune animalsbecause the antibody can be easily prepared. For immunization, it ispreferable from the standpoint of inducing antibody production to useFreund's complete adjuvant or another auxiliary to prepare an emulsionwhich is then administered multiple times. In addition to Freund'scomplete adjuvant (FCA), Freund's incomplete adjuvant (FIA), ammoniumhydroxide gel and the like can be used as auxiliaries. The antigendosage per individual mammal can be determined based on the kind ofmammal, but in the case of mice it is ordinarily 50-500 μg.Administration may be intravenous, subcutaneous, intraperitoneal or thelike. Immunization is normally performed at intervals of between severaldays and several weeks, preferably at intervals of 4 days to 3 weeks fora total of 2-8 or preferably 2-5 immunizations. 3-10 days after the lastimmunization, antibody titer for protein (a) or (b) is measured, andafter antibody titer rises blood is taken and antiserum obtained.Antibody titer can be measured by enzyme-linked immunosorbent assay(ELISA), radio-immuno-assay (RIA) or the like.

[0091] When antibodies need to be purified from the antiserum, a wellknown method such as salting out with ammonium sulfate, gelchromatography, ion exchange chromatography, affinity chromatography orthe like or a combination of such methods can be selected asappropriate.

[0092] To prepare monoclonal antibodies, mammals are immunized usingimmune antigen as in the case of polyclonal antibodies, andantibody-producing cells are harvested 2-5 days after the finalimmunization. Examples of antibody-producing cells include spleen cells,lymph node cells, thymus cells, peripheral blood cells and the like, butspleen cells are normally used.

[0093] Next, cell fusion of the antibody-producing cells with myelomacells is performed to obtain a hybridoma. Commonly available cellsstrains derived from humans, mice or other mammals can be used as themyeloma cells for fusion with the antibody-producing cells. It ispreferable that the cell strain used be one that has drug selectivityand that does not survive in unfused form in selection medium (such asHAT medium), but which only survives when fused with theantibody-producing cells. Specific examples of myeloma cells includeP3X63-Ag.8.U1 (P3U1), P3/NSI/1-Ag4-1, Sp2/0-Ag14 and other mouse myelomacell strains.

[0094] Cell fusion is performed by mixing specific proportions (such asbetween 1:1 and 1:10) of the antibody-producing cells and myeloma cellsin an animal cell culture medium such as DMEM or RPMI-1640 whichcontains no serum, and then performing a fusion reaction withpolyethylene glycol or another cell fusion accelerator or by electricalpulse treatment (such as electroporation).

[0095] Following cell fusion treatment, culture is performed in aselection medium, and the target hybridoma selected. Next, the culturesupernatant of the multiplied hybridoma is screened to confirm thepresence of the target antibodies. Hybridoma screening may be accordingto ordinary methods without any particular limitations. For example,part of the culture supernatant in a well containing a hybridoma can beharvested and screened by enzyme-linked immunosorbent assay (ELISA),radio-immuno-assay (RIA) or the like.

[0096] The hybridoma can be cloned by limiting dilution analysis, softagar cloning, fibrin gel cloning, fluorescence activated cell sortingand the like, to finally obtain a hybridoma which produces monoclonalantibodies.

[0097] Harvesting of monoclonal antibodies from the resulting hybridomacan be accomplished by an ordinary cell culture method or the like. Inthe cell culture method, the hybridoma is cultured for example for 3-10days under ordinary culture conditions (such as 37° C., 5% CO₂) in ananimal cell culture medium such as MEM medium or RPMI-1640 mediumcontaining 10-20% fetal calf serum, and monoclonal antibodies areobtained from the culture supernatant. The hybridoma can also betransplanted intraperitoneally to mice or the like, ascites collectedafter 10-14 days, and monoclonal antibodies obtained from the ascites.

[0098] When monoclonal antibodies need to be purified, a well-knownmethod such as salting out with ammonium sulfate, gel chromatography,ion exchange chromatography, affinity chromatography or the like or acombination of such methods can be selected as appropriate.

[0099] When monoclonal antibodies are intended for administration tohuman beings (antibody therapy), human antibodies or humanizedantibodies should be used to reduce immunogenicity. Human antibodies orhumanized antibodies are obtained for example by preparing a hybridomausing as the immune animals mice or the like having introduced humanantibody DNA, or by using a library having the antibodies presented on aphage. Specifically, a transgenic animal having a repertory of humanantibody DNA is immunized with the antigen as a protein orprotein-expressing cells or solution thereof to obtainantibody-producing cells, which are then fused with myeloma cells toproduce a hybridoma which is used to obtain human antibodies to thetarget protein (see International Publication Nos. WO92-03918,WO93-2227, WO94-02602, WO96-33735 and WO96-34096). Alternatively, scFvwhich binds to the target protein can be selected by sorting out, froman antibody library having multiple different human scFv presented onphages, those phages presenting antibodies which bind to the antigen asa protein or protein-expressing cells or solution thereof (Griffiths etal, EMBO J. 12, 725-734, 1993).

[0100] The screening method of the present invention comprises a step ofbringing a test substance into contact with protein (a) or (b). In thescreening method of the present invention, a ligand, agonist orantagonist to an adiponectin receptor can be screened by bringing a testsubstance into contact with protein (a) or (b) and determining whetherthe test substance binds to protein (a) or (b). The screened substancesshould also be subjected to another step to determined whether or notthey actually act as a ligand, agonist or antagonist to an adiponectinreceptor.

[0101] There are no particular limits on the types of test substances,but examples include high molecular compounds, low molecular compounds,cell cultures, tissue extracts and the like.

[0102] For screening purposes, (i) cells or tissues or processed cellsor tissues expressing protein (a) or (b), (ii) recombinant proteinsexpressed by introduction of DNA encoding protein (a) or (b) into E.coli, yeast, insect cells, animal cells or another host by recombinanttechnology, or (iii) chemically synthesized peptides for example can beused as protein (a) or (b). The cells or tissues expressing protein (a)or (b) may be either cells or tissues (such as muscle cells, skeletalmuscles, hearts, macrophages, blood vessels, brains, kidneys, lungs,placentas, spleens, testes, peripheral blood, thymus glands, intestinesor the like) which express protein (a) or (b) as an endogenous protein,or cells or tissues which express protein (a) or (b) as an exogenousprotein (such as E. coli, yeast, insect cells, animal cells or the likehaving introduced DNA encoding protein (a) or (b)). Processed cells ortissues are cells are tissues which have been crushed, extracted,purified or the like, and include for example cell membrane fractions ofcells or tisues.

[0103] Whether or not the test substance acts as a ligand, agonist orantagonist to the adiponectin receptor can be determined for examplebased on the amount of binding of the test substance to protein (a) or(b) or according to the presence or degree of cell response due tobinding of the test substance to protein (a) or (b).

[0104] The amount of binding of the test substance to protein (a) or (b)can be measured for example using a labeled test substance, labeledantibodies to the test substance or the like. Radioactive isotopeelements such as ³H, ¹⁴C, ¹²⁵I, ³⁵S, and ³²P and fluorescent dyes andthe like can be used as labels. The radioactivity of the radioactiveisotope element can be measured using a liquid scintillation counter,X-ray film, imaging plate or the like, while the fluorescent strength ofthe fluorescent dye can be measured for example by a CCD camera,fluorescent scanner, spectrofluorometer or the like.

[0105] Examples of cell responses due to binding of the test substanceto protein (a) or (b) include stimulation or suppression of PPARα ligandactivity, stimulation or suppression of fatty acid oxidation,stimulation or suppression of glucose uptake, increased or decreasedintracellular pH, increased or decreased AMP kinase activity, increasedor decreased AMP kinase phosphorylation, increased or decreased p38 MAPkinase activity, increased or decreased p38 MAP kinase phosphorylation,stimulation or suppression of gluconeogenesis, and increased ordecreased uncoupled protein and the like.

[0106] In measuring the amount of binding of the test substance toprotein (a) or (b) and the presence or degree of cell response, it isdesirable to compare measurement values with and without protein (a) or(b).

[0107] Lipid cells secrete adiponectin as a vital insulin sensitizinghormone, and lipid cell hypertrophy leads to decreased adiponectinexcretion, which leads to insulin resistance, which in turn is a causeof diabetes, hyperlipidemia and hypertension. Decreased adiponectinexcretion also promotes arteriosclerosis. Consequently, substances whichhave been screened as an agonist or antagonist to an adiponectinreceptor can be used for example as insulin resistance improving drugs,diabetes preventive and therapeutic drugs, hyperlipidemia preventive andtherapeutic drugs, hypertension preventive and therapeutic drugs,arteriosclerosis preventive and therapeutic drugs, obesity preventiveand therapeutic drugs, anti-inflammatory drugs, osteoporosis preventiveand therapeutic drugs, anti-cancer drugs and the like.

[0108] These drugs may be composed solely of the screened substances,but normally they are prepared using pharmaceutically acceptableexcipients and other additives as desired. For purposes of preparation,excipients, binders, disintegrators, lubricants, stabilizers, flavoringsand perfumes, diluents, solvents for injection and other additives canbe used for example. Administration may be oral or parenteral(subcutaneous, intramuscular, intraperitoneal or the like) for example,and forms of administration include sprays, capsules, pills, granules,syrups, emulsions, suppositories, injections, suspensions and the like.The dosage and number of administrations will differ according to thedesired effects, administration method, treatment period, and age,weight, sex and the like of the patient, and can be adjustedappropriately according to the type of screened substance.

[0109] Protein (a) or (b), a gene encoding protein (a) or (b), arecombinant vector containing the DNA and a transformant containing therecombinant vector can be used as constituent elements in a screeningkit for a ligand, agonist and antagonist to an adiponectin receptor.They are included in the screening kit as suppliers of protein (a) or(b).

[0110] The screening kit can take any form as long as it includesprotein (a) or (b), a gene encoding protein (a) or (b), a recombinantvector containing the DNA or a transformant containing the recombinantvector, and can include various reagents (buffers and the like),measurement equipment, labeling compounds, model animals, cell strains,cell culture media and the like.

EXAMPLES

[0111] In the following text and figures relating to the examples,adiponectin may be referred to as “Adipo,” adiponectin consisting solelyof the globular domain as “globular Adipo” or “gAd,” and full-lengthadiponectin as “full-length Adipo” or “Ad.” An adiponectin receptor maybe referred to as “AdipoR,” a human adiponectin receptor as “hAdipoR,”and a mouse adiponectin receptor as “mAdipoR”. “AdipoR1” and “AdipoR2”are designations for adiponectin receptors comprising different aminoacid sequences.

[0112] 1. Experimental Methods

[0113] (1) Retrovirus Production and Infection

[0114] For production of retroviral supernatants, 10⁷ Plat-E packagingcells (Morita, S. et al, Gene Ther. 7, 1063-1066 (2000)) weretransiently transfected with 10 μg of human skeletal muscle cDNA library(Clontech) using Lipofectamine PLUS (Life Technologies). After 24 hoursof incubation the supernatants (10 mL) was harvested. Ba/F3 cells wereinfected with 1/20-diluted supernatants supplemented with 10 μg/mLpolybrene (hexadimethrine bromide, Sigma) corresponding to an estimatedm.o.i. of 0.3. Six hours later, the medium was changed, and the Ba/F3cells were expanded by culturing for 6 days prior to freezing orselection.

[0115] (2) FACS Analysis and cDNA Sequencing

[0116] FACS analysis was performed according to the method of Stoecklinet al (Stoecklin, G. et al, EMBO J. 21, 4709-4718 (2000)). Forselection, cells were enriched with FACVantage (Becton Dickinson) from1×10⁷ Ba/F3 cells transfected with human skeletal muscle cDNA library.The cells were recovered, expanded, and subjected to FACS analysis 11days later. The selected cells were further expanded and subjected toFACS analysis. In order to sequence the cDNA incorporated into theselected cells, PCR was performed with upstream and downstream primersfor the retrovirus vector, using 50 ng of genome DNA extracted from theselected cells as the template, and the resulting PCR amplifiedfragments were sequenced.

[0117] PCR was performed in 35 cycles of 1 minute at 94° C., 2 minutesat 56° C. and 3 minutes at 72° C., using Taq polymerase(Perkin-Elmer/Cetus). The primers (pLIB primers) for the retrovirusvector were as follows: 5′ primer: 5′-agccctcactccttctctag-3′ 3′ primer:5′-acctacaggtggggtctttcattccc-3′

[0118] After removal of the primer, the base sequence of the PCR productwas determined by direct sequencing using a BigDye Terminator Kit(Applied Biosystems).

[0119] (3) Northern Blot Analysis

[0120] Human multiple tissues Northern blot filters I and II (brain,heart, skeletal muscle, colon, thymus, spleen, kidney, liver, smallintestine, placenta, lung, peripheral blood leukocytes) were purchasedfrom Clonetech. These filters were hybridized with [³²P] dCTP-labeledcDNA probes (the PstI/BstXI, BamHI/PstI and EcORV/NotI fragment of humanAdipoR1 cDNA, mouse AdipoR1 cDNA, and human and mouse AdipoR2 cDNA,respectively) in a hybridization buffer containing 4×SSC, 5× Denhardt'ssolution, 0.2% SDS, 200 mg/mL salmon sperm DNA and 50% formamide at 42°C. for 24 hours. The filters were washed in 0.1×SSC, 0.1% SDS at 65° C.,and then subjected to autoradiography. The same northern blot analysiswas performed on various mouse tissues (brain, heart, kidney, liver,lung, skeletal muscle, spleen, testis).

[0121] (4) Expression of Proteins in Mammalian Cells, andCharacterization

[0122] The AdipoR1 or R2 expression vector was constructed by ligatingAdipoR1 or AdipoR2 cDNA into the EcORV/NotI site of pCXN2 (Kinoshita, S.et al, Pharm Res. 15, 1851-1856 (1998)). HEK-293T (human embryonickidney cells), HAEC (normal human aortic endothelial cells) and C2C12myocytes (mouse myocyte strain) were cultured in DMEM. 10% fetal calfserum (FCS) was contained in the medium. DNA transfection was performedby lipofection using Lipofectamine PLUS (Gibco BRL) for all cells.

[0123] (5) RNA Interference in C2C12 Myocytes

[0124] According to the method of Karpichev et al (Karpichev, I. V., J.Biol. Chem. 277, 19609-19617 (2002)), two pairs of siRNA were chemicallysynthesized, annealed, and transfected into C2C12 myocytes which hadbeen differentiated into myotube cells by 4-7 days culture in DMEMcontaining horse serum after 2 days from confluence. siRNA was alsotransfected into hepatocytes and HAEC in the same way. 48 hours aftertransfection of siRNA, the cells were lysed.

[0125] The base sequences of the unrelated control siRNA (siRNA) are asfollows: Unrelated-sense: gugcgcugcuggugccaaccctt Unrelated-antisense:ggguuggcaccagcagcgcactt

[0126] The base sequences of siRNA (siRNA mAdipoR1) corresponding to thecoding region of the mouse AdipoR1 gene are as follows: siRNAmAdipoR1-sense: gagacuggcaacaucuggacatt siRNA mAdipoR1-antisense:uguccagauguugccagucuctt

[0127] The base sequences of siRNA (siRNA mAdipoR2) corresponding to thecoding region of the mouse AdipoR2 gene are as follows: siRNAmAdipoR2-sense: gcuuagagacaccuguuuguutt siRNA mAdipoR2-antisense:aacaaacaggugucucuaagctt

[0128] The base sequences of siRNA (siRNA hAdipoR1) corresponding to thecoding regions of the human AdipoR1 gene are as follows: siRNAhAdipoR1-sense: ggacaacgacuaucugcuacatt siRNA hAdipoR1-antisense:uguagcagauagucguugucctt

[0129] The base sequences of siRNA (siRNA hAdipoR2) corresponding to thecoding regions of the human AdipoR2 gene are as follows: siRNAhAdipoR2-sense: ggaguuucguuucaugaucggtt siRNA hAdipoR2-antisense:ccgaucaugaaacgaaacucctt

[0130] (6) Measurement of PPARα (Peroxisome Proliferator-ActivatedReceptor α) Ligand Activity

[0131] Mouse globular Adipo and full-length Adipo expressed using E.coli were purified according to the method of Yamauchi et al (Yamauchi,T. et al, Nat. Med. 8, 1288-1295 (2002)). According to the method ofYamauchi et al al (Yamauchi, T. et al, Nat. Med. 8, 1288-1295 (2002)),the differentiated C2C12 myocytes or isolated hepatocytes were treatedwith a fixed concentration of adiponectin. PPARα ligand activity wasquantitatively determined according to the method of Yamauchi et al(Yamauchi, T. et al, J. Biol. Chem. 278, 2461-2468 (2002)) using a (UAS)X 4-tk-LUC reporter plasmid, a GAL4-rat PPARα ligand binding domainexpressing plasmid and a β-galactosidase expressing plasmid (internalcontrol).

[0132] (7) Lipid and Glucose Metabolism

[0133] [¹⁴C] CO₂ production from [¹⁻¹⁴C] palmitic acid was measuredusing cell lysate according to the method of Yamauchi et al (Yamauchi,T. et al, Nat. Med. 7, 941-946 (2001)). Glucose uptake was also measuredaccording to the method of Yamauchi et al (Yamauchi, T. et al, Nat. Med.8, 1288-1295 (2002)).

[0134] (8) Study Using Dominant Negative AMP Kinase (AMPK)

[0135] cDNA encoding α2 AMPK (including a mutation that alters lysineresidue #45 to arginine residue) was used as DN-α2 AMPK (Yamauchi, T. etal, Nat. Med. 8, 1288-1295 (2002)). C2C12 myocytes were infected withequal titers of adenovirus containing a control MOCK vector or DN-α2AMPK. According to the method of Yamauchi et al (Yamauchi, T. et al,Nat. Med. 8, 1288-1295 (2002)), 5 days after induction ofdifferentiation, the cells were treated with a fixed concentration ofAdipo, and PPARα ligand activity and fatty acid oxidation were measured.

[0136] (9) Binding Assay

[0137] Synthetic human or mouse Adipo was [¹²⁵]-labeled at Tyr withIODO-beads (Pierce) in the presence of Na¹²⁵I (2000 Ci/mmol, AmershamPharmacia Biotech). Recombinant globular Adipo or full-length Adipo wasbiotinylated with NHS-LC-Biotin (Pierce). The cells were seeded on96-well plates at a density of 4.1×10⁴/well. After an overnight culture,the medium was discarded and the cells were incubated overnight at 37°C. with a binding assay buffer (HEPES buffered saline/0.1% bovine serumalbumin) containing fixed concentrations of [¹²⁵I] Adipo and unlabeledcompetitors. According to the methods of Yamauchi et al (Yamauchi, T. etal, Nat. Med. 8, 1288-1295 (2002)); Yokomizo, T. et al, Nature 387,620-624 (1997)), the cells were then washed three times with ice-coldphosphate-buffered saline and lysed in 0.1N NaOH/0.1% SDS, and thecell-bound radioactivity was determined using a γ-counter.

[0138] (10) Fluorescent Microscopic Analysis

[0139] The cellular location of AdipoR1 or AdipoR2 was evaluated byconfocal fluorescence microscopy using 293T cells. The cells were fixedin 1% paraformaldehyde, with or without permeabilization using 5×diluted permeabilization buffer (Coulter), and the cells were incubatedfor 1 hour at 22° C. with anti-FLAG antibody (M2; 30 μg/mL). Then theywere incubated with 10 μg/mL of secondary antibody conjugated toAlexFluor 488. Confocal imaging was then performed with a laser-scanningmicroscopy system configured with a Nikon microscope and a Krypton/argonlaser (488 nm).

[0140] (11) Quantitative Analysis of AdipoR1 and AdipoR2 GeneTranscripts by Real-Time PCR

[0141] Quantitative analysis of four transcripts corresponding to thehuman AdipoR1, human AdipoR2, mouse AdipoR1 and mouse AdipoR2 genes wasperformed by real-time PCR according to the method of Heid et al (Heid,C. A. et al, Genome Res. 6, 986-994 (1996)). The primer sets and theprobes for the transcripts were as follows. The PCR product was measuredcontinuously using an ABI PRISM7700 Sequence Detection System (AppliedBiosystems). The relative amounts of each transcript were normalized tothe amount of actin transcript. [Mouse AdipoR1 gene] Forward primer:5′-acgttggagagtcatcccgtat-3′ Reverse primer: 5′-ctctgtgtggatgcggaagat-3′Probe: 5′-cctgctacatggccacagaccacct-3′ (with minor groove binder) [MouseAdipoR2 gene] Forward primer: 5′-tcccaggaagatgaagggtttat-3′ Reverseprimer: 5′-ttccattcgttcgatagcatga-3′ Probe: 5′-atgtccccgctcctacaggccc-3′(with minor groove binder) [Human AdipoR1 gene] Forward primer:5′-ttcttcctcatggctgtgatgt-3′ Reverse primer: 5′-aagaagcgctcaggaattcg-3′Probe: 5′-tcactggagctggcctttatgctgc-3′ (with minor groove binder) [HumanAdipoR2 gene] Forward primer: 5′-atagggcagataggctggttga-3′ Reverseprimer: 5′-ggatccgggcagcataca-3′ Probe:5′-ctgatggccagcctctacatcacagga-3′ (with minor groove binder)

[0142] (12) Measurement of Intracellular Calcium Concentration, and cAMPand cGMP Levels

[0143] Intracellular Ca²⁺ concentrations were measured according to themethod of Yokomizo et al (Yokomizo, T. et al, Nature 387, 620-624(1997)). Namely, 10 μM of Fura-2/AM (Dojin) dissolved in Hepes-Tyrode'sBSA buffer (25 mM Hepes-NaOH (pH 7.4), 140 mM NaCl, 2.7 mM KCl, 1.0 mMCaCl₂ 12 mM NaHCO₃, 5.6 mM D-glucose, 0.37 mM NaH₂PO₄, 0.49 mM MgCl₂,0.1% [wt/vol] BSA without fatty acids; Fraction V) was contacted withthe cells at 37° C. for 2 hours. The cells were washed twice, andsuspended at a concentration of 10⁶ cells/mL in Hepes-Tyrode's BSAbuffer. 0.5 mL of cell suspension was applied to a CAF-100 system(Jasco), and 5 μL of ligand ethanol solution (for LTB4) or PBS solution(for Adipo) added. Intracellular Ca²⁺ concentrations were measured basedon the proportions of 500 nm fluorescence generated by excitation lightat 340 and 380 nm.

[0144] cAMP and cGMP levels were measured according to the method ofYokomizo et al (Yokomizo, T. et al, Nature 387, 620-624 (1997)) usingassay kits (Biotrak cAMP EIA System for cAMP, Biotrak cGMP EIA Systemfor cGMP, Amersham Pharmacia Biotech) in accordance with manufacturer'sprotocols.

[0145] (13) Predicted Structures of AdipoR1 and AdipoR2

[0146] Hydropathy plots of the AdipoR1 and AdipoR2 proteins wereconducted using the hydrophobicity indices of Kyte-Doolittle. Structuralmodels for AdipoR1 and AdipoR2 were also predicted by SOSUI, andconsensus sequences analyzed by PRINTS(http://bioinf.man.ac.uk/dbbrowser/PRINTS/). In addition, thephosphorylation site was analyzed with DNASIS Pro. Finally, the methoddescribed at http://cbrg.inf.ethz.ch/Server/AllAll.html was used toanalyze whether AdipoR1/R2 have homology to any other class of GPCR.

[0147] (14) Phosphorylation and Amount of Phosphorylation of AMP Kinase(AMPK), ACC, p38 MAP Kinase (p38 MAPK) and MAP Kinase (MAPK)

[0148] Phosphorylation and amount of phosphorylation of AMPK, ACC(Yamauchi, T. et al, Nat. Med. 8, 1288-1295 (2002)), p38 MAPK and MAPK(Barger, P. M. et al, J. Biol. Chem. 276, 44495-44501 (2001);Puigserver, P. et al, Mol. Cell 8, 971-982 (2001); Michael, L. F. et al,Proc. Natl. Acad. Sci. USA 98, 3820-3825 (2001)) were measured bywestern blotting using anti-phosphorylated AMPK antibodies,anti-phosphorylated ACC antibodies, anti-phosphorylated p38 MAPKantibodies and anti-phosphorylated MAPK antibodies. In this test, C2C12cells or hepatocytes transfected or not transfected with AdipoR1 wereincubated for 10 minutes with 0.1 μg/mL or 1 μg/mL gAd, and lysates ofthe various cells were reacted with their respective antibodies.

[0149] 2. Results

[0150] (1) Expression Cloning of AdipoR1 and AdipoR2

[0151] In muscle, globular Adipo ameliorates insulin resistance andstimulates PPARα and fatty acid oxidation more than does full-lengthAdipo (Fruebis, J. et al, Proc. Natl. Acad. Sci. USA 98, 2005-2010(2001); Yamauchi, T. et al, Nat. Med. 7, 941-946 (2001); Yamauchi, T. etal, J. Biol. Chem. 278, 2461-2468 (2002)). Moreover, globular Adipobinds more strongly to C2C12 cells than does full-length Adipo, and alsobinds more strongly to skeletal muscle membranes than to hepatocytes andliver membranes (Yamauchi, T. et al, Nat. Med. 8, 1288-1295 (2002)) (seeFIGS. 1a and 1 b). FIG. 1a shows binding of globular Adipo orfull-length Adipo to C2C12 myocytes, while FIG. 1b shows binding ofglobular Adipo or full-length Adipo to hepatocytes. The cells wereincubated with biotinylated globular Adipo or full-length Adipo at theconcentrations shown, and biotinylated globular Adipo or full-lengthAdipo bound to the cell surfaces was assayed by ELISA. The bars in thefigures indicate mean±s.e. (n=3-5), with “*” indicating P<0.05 and “**”P<0.01.

[0152] An effort was then made to isolate AdipoR1 cDNA by screeningproteins with globular Adipo binding ability from a library prepared byinfecting Ba/F3 cells with a retrovirus having incorporated cDNA derivedfrom human skeletal muscle mRNA.

[0153] The infected Ba/F3 cells were collected, incubated withbiotinylated globular Adipo, stained with streptavidin-conjugatedphycoerythrin (PE; red fluorescent probe), and subjected tofluorescence-activated cell sorting (FACS) (see FIG. 1c). The results ofFACS analysis are shown in FIGS. 1 c, 1 d and 1 e. FIG. 1c shows theBa/F3 cells after staining but before first sorting, FIG. 1d shows theBa/F3 cells before third sorting, and FIG. 1e shows the Ba/F3 cellsafter incubation with globular Adipo bound with FITC (fluoresceinisothiocyanate) but before fourth sorting. The regions in rectangularboxes in FIGS. 1c, 1 d and 1 e indicate AdipoR1-positive cells, withthose in the boxes being selected cells.

[0154] The cells selected for globular Adipo binding ability were thensubjected to a second round of sorting. The resorted cells that werepositive for globular Adipo binding ability (see FIG. 1d) were subjectedto a third round of sorting, and the resorted cells immediatelyincubated with globular Adipo conjugated with FITC (fluoresceinisothiocyanate; green fluorescent probe). Since cells which changed fromred to green were those having specific binding sites for globular Adipo(see FIG. 1e), only those cells which changed in this way were selectedand cultured to expand them for further analysis. Genomic DNA extractedfrom these cells was subjected to PCR using viral vector primers, andsequenced.

[0155] The cells were stained in two colors (red and green) as describedabove in order to eliminate cells to which globular Adipo was boundnon-specifically by its adhesiveness.

[0156] The base sequence of the sequenced human AdipoR1 cDNA is shown bySeq. No. 1, and the amino acid sequence of the protein (human AdipoR1)encoded thereby by Seq. No. 2. The base sequence of mouse AdipoR1 cDNAobtained by similar methods from C2C12 myocytes is shown by Seq. No. 5,and the amino acid sequence of the protein (mouse AdipoR1) encodedthereby by Seq. No. 6. Human AdipoR1 cDNA (Seq. No. 1) and mouse AdipoR1cDNA (Seq. No. 5) are shown to be genes encoding proteins consisting of375 amino acids (see FIG. 1f). There is 96.8% homology between the aminoacid sequences of human AdipoR1 and mouse AdipoR1 (see FIG. 1f). FIG. 1fshows model structures of AdipoR1 and AdipoR2 gene transcripts indatabases (NIH-MGC Project and NCBI contig).

[0157] From previous findings (Yamauchi, T. et al, Nat. Med. 8,1288-1295 (2002); Yamauchi, T. et al, J. Biol. Chem. 278, 2461-2468(2002)), it appears that there are two types of AdipoR with distinctbinding affinities for globular Adipo or full-length Adipo, one typewhich preferentially binds globular Adipo expressed in skeletal muscle,and another which binds only full-length Adipo expressed in liver.

[0158] A search for proteins that share homology with AdipoR1 led to thediscovery in human and mouse databases (The Human Genome,http://www.ncbi.nlm.nih.gov/genome/guide/human/; Mouse Genome Resources,http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) (Waterston, R. H. etal, Nature 420, 520-562 (2002); Okazaki, Y. et al, Nature 420, 563-573(2002)) of a gene having an open reading frame (ORF) different from thatof AdipoR1 cDNA. This cDNA was cloned from the mRNA of HepG2 cells (cellstrain derived from human liver cancer) and sequenced, and the proteinencoded by this cDNA was named AdipoR2. The base sequence of thesequenced human AdipoR2 cDNA is shown by Seq. No. 3, and the amino acidsequence of the protein encoded thereby (human AdipoR2) by Seq. No. 4.Furthermore, the base sequence of mouse AdipoR2 cDNA obtained by thesame methods from C2C12 myocytes is shown by Seq. No. 7, and the aminoacid sequence of the protein encoded thereby (mouse AdipoR2) by Seq. No.8.

[0159] There is 95.2% homology between the amino acid sequences of humanAdipoR2 and mouse AdipoR2. AdipoR1 and AdipoR2 are extremely similarstructurally, with 66.7% homology between the amino acid sequences ofmouse AdipoR1 and AdipoR2.

[0160] In SWISS-PROT, there were no mammalian proteins having highhomology with AdipoR1 and AdipoR2, but interestingly the cDNA encodingAdipoR1 and AdipoR2 had homology with yeast YOL002c (Karpichev, I. V. etal, J. Biol. Chem. 277, 19609-19617 (2002)). YOL002c is reported toencode a seven transmembrane protein that plays a key role in metabolicpathways which regulate lipid metabolism such as fatty acid oxidation(Karpichev, I. V. et al, J. Biol. Chem. 277, 19609-19617 (2002)). Itappears that AdipoR/YOL002c with a seven transmembrane structure acrossspecies mediates key regulatory signals in lipid metabolism such asfatty acid oxidation.

[0161] (2) Tissue Distribution of AdipoR1 and AdipoR2

[0162] The results of northern blot analysis of various mouse tissuesare shown in FIG. 1g, and the results of northern blot analysis ofvarious human tissues in FIG. 1h. In FIG. 1g, lane 1 shows results forthe brain, lane 2 for heart, lane 3 for kidney, lane 4 for liver, lane 5for lung, lane 6 for skeletal muscle, lane 7 for spleen, and lane 8 fortestis, while in FIG. 1h, lane 1 shows results for brain, lane 2 forheart, lane 3 for skeletal muscle, lane 4 for colon, lane 5 for thymus,lane 6 for spleen, lane 7 for kidney, lane 8 for liver, lane 9 for smallintestine, lane 10 for placenta, lane 11 for lung and lane 12 forperipheral blood leukocytes.

[0163] Northern blot analysis of various human and mouse tissues,identified one major 2.0 kb band with the predicted mRNA size in theaforementioned database, and also revealed that AdipoR1 is expressed inmost tissues and most abundantly expressed in skeletal muscle. One major4.0 kb band with the predicted mRNA size was also identified in theaforementioned database, and it was shown that AdipoR2 is mostabundantly expressed in the liver.

[0164] (3) Cellular Locations of AdipoR1 and AdipoR2

[0165] From the deduced amino acid sequence (Seq. No. 6) of mouseAdipoR1, it is predicted that mouse AdipoR1 is a protein consisting of375 amino acids and having a molecular weight of 42.4 kDa. From thededuced amino acid sequence (Seq. No. 8) of mouse AdipoR2, it ispredicted that mouse AdipoR2 is a protein consisting of 311 amino acidsand having a molecular weight of 35.4 kDa (see FIG. 2a). In FIG. 2a,which shows the results of a scan of the AdipoR sequence by PRINTSsoftware (http://bioinf.man.ac.uk/dbbrowser/PRINTS/), the underlinedregions are the 7 transmembrane domains of AdipoR1 and AdipoR2, whilethe areas underlined in bold indicate the characteristic conservedmotifs of G-protein coupled receptor members. PKC phosphorylation sitesand tyrosine phosphorylation sites are also shown in FIG. 2a.

[0166] From the deduced amino acid sequences of AdipoR1 and AdipoR2, itis predicted that AdipoR1 and AdipoR2 are proteins having 7transmembrane domains (see FIG. 2a). An alignment of AdipoR1 and AdipoR2was performed with known receptors having 7 transmembrane domains(Waterston, R. H. et al, Nature 420, 520-562 (2002); Okazaki, Y. et al,Nature 420, 563-573 (2002); Wess, J., FASE B. J. 11, 346-354 (1997)),but homology to the amino acid sequences of members of the G-proteincoupled receptor (GPCR) family was low. AdipoR1 and AdipoR2 lackedcharacteristics (such as conserved amino acids, glycosylation sites,sites for G-protein coupling) of the G-protein coupled receptor family(Wess, J. et al, FASEB. J. 11, 346-354 (1997); Yokomizo, T. et al,Nature 387, 620-624 (1997); Scheer, A. et al, EMBO. J. 15, 3566-3578(1996)). Of the highly conserved amino acids in the G-protein coupledreceptor family, only one of two highly conserved Cys residues werepresent in the first and second extracellular loops of AdipoR1 andAdipoR2. AdipoR1 and R2 lacked the highly conserved Asn-Pro-Xaa2-Tyrmotif present at the end of TM7. AdipoR1 and AdipoR2 also lacked thehighly conserved Asp-Arg-Tyr motif present at the TM3/intracellular loop2 transition (Wess, J. et al, FASEB. J. 11, 346-354 (1997); Scheer, A.et al, EMBO. J. 15, 3566-3578 (1996)).

[0167] Human and mouse AdipoR1 or AdipoR2 labeled with epitope tag FLAGwas expressed in HEK-293 cells, and immunoblotted with anti-FLAGantibodies. Human and mouse AdipoR1 and AdipoR2 exhibited the expectedmolecular weights (see FIG. 2b).

[0168] To determine the subcellular location and topology of mouseAdipoR1 and AdipoR2, AdipoR1 or AdipoR2 cDNA with epitope tags at eitherend was expressed in HEK-293T cells (see FIG. 2c). In FIG. 2c, “intact”indicates that the cells were not permeabilized and “permeabilized”indicates that they were.

[0169] When the epitope tag was inserted at the N-terminus, AdipoR1 andAdipoR2 could be detected at the cell surface only when the cells werepermeabilized (see FIG. 2c). In contrast, when the epitope tag wasinserted at the C-terminus, AdipoR1 and AdipoR2 could be detected at thecell surface (see FIG. 2c). These results indicate that AdipoR1 andAdipoR2 are integral membrane proteins with seven transmembrane domains,in which the N-terminus is within the membrane and the C-terminus isoutside the membrane (see FIGS. 2c, 2 d). This is opposite to thetopology of all reported G-protein coupled receptors (Wess, J. et al,FASEB. J. 11, 346-354 (1997); Yokomizo, T. et al, Nature 381, 620-624(1997); Scheer, A. et al, EMBO J. 15, 3566-3578 (1996)). Hypotheticalstructural models of AdipoR1 and AdipoR2 are shown in FIGS. 2d and 2 e,respectively.

[0170] (4) Effects of AdipoR Expression in 293T Cells

[0171] The binding and intracellular signaling stimulated by globularAdipo or full-length Adipo using 293T cells overexpressing AdipoR1 orAdipoR2 on the cell surface were examined. Expression of AdipoR1 orAdipoR2 in 293T cells enhanced binding of both globular Adipo andfull-length Adipo (see FIGS. 3a, 3 b). FIG. 3a shows the bindingisotherm of [¹²⁵I] globular Adipo (gAd) binding to 293T cellstransfected with AdipoR1 or AdipoR2, while FIG. 3b shows the bindingisotherm of [¹²⁵I] full-length Adipo (Ad) binding to the 293T cells. Inthese figures, a white square indicates a result for mock, a blacksquare a result for mouse AdipoR1 and a black circle a result for mouseAdipoR2.

[0172] We next examined whether AdipoR1 and AdipoR2 could formmultimers. When AdipoR1 with the epitope tag FLAG and AdipoR1 with theepitope tag HA were co-expressed in HEK-293T cells, AdipoR1 with theepitope tag FLAG was detected in anti-HA antibody immunoprecipitates(see FIG. 3c). FIG. 3c shows the results when cell lysates of 293T cellstransfected with AdipoR1 or AdipoR2 having epitope tag FLAG or HA wereimmunoprecipitated (IP) with anti-FLAG antibody (upper and lowerpanels), and then immunoblotted with anti-FLAG (upper panel) or anti-HA(lower panel) antibody, and indicates formation of AdipoR1 and AdipoR2homo- and hetero-multimers.

[0173] Moreover, when AdipoR1 with the epitope tag HA and AdipoR2 withthe epitope tag FLAG were co-expressed in HEK-293T cells, AdipoR2 withthe epitope tag FLAG was detected in anti-HA antibody immunoprecipitates(see FIG. 3c). These data suggest that AdipoR1 and AdipoR2 may be ableto form both homo- and hetero-multimers.

[0174] In cells expressing AdipoR1, Adipo had no apparent effect onintracellular calcium, although LTB4 increased intracellular calcium incells expressing either GPCR or LTB4 receptor BLT1 (see FIG. 3d), andthese cells showed similar expression levels to those of cellsexpressing AdipoR1 (data not shown). FIG. 3d shows measurement resultsfor changes in [Ca²⁺ ]i when BLT1-expressing cells or mouseAdipoR1-expressing cells loaded with Fura-2/AM were challenged with 10μg/mL globular Adipo (gAd), 10 μg/mL full-length Adipo (Ad), 1 μM LTB4or 100 μM ATP.

[0175] Moreover, in cells expressing AdipoR1, AdipoR1 had little or noapparent effect on cAMP and cGMP levels (see FIG. 3e). FIG. 3e showsresults for accumulation of cAMP or CGMP in HEK-293 cells treated withor without forskolin, with “gAd0.01” and “gAd0.1” indicating 0.01 or 0.1μg/mL of globular Adipo, respectively, and “Ad1” and “Ad10” indicating 1and 10 μg/mL of full-length Adipo, respectively. In contrast, expressionof AdipoR1 enhanced increases in PPARα ligand activity by globular Adipoand full-length Adipo in 293T cells (see FIG. 3f). FIG. 3f shows inPPARα ligand activity in AdipoR1-transfected 293T cells incubated withthe indicated concentrations (μg/mL) of globular Adipo or full-lengthAdipo, with “gAd0.1,” “gAd0.5” and “gAd2.5” indicating 0.1, 0.5 and 2.5μg/mL of globular Adipo, respectively, and “Ad1,” “Ad5” and “Ad25”indicating 1, 5 and 25 μg/mL of full-length Adipo, respectively. In thefigure each bar shows mean±s.e. (n=3-5), with indicating P<0.05 and “**”P<0.01.

[0176] (5) PPARα Activation and Fatty Acid Oxidation in C2C12 Myocytes

[0177] Expression of AdipoR1 in C2C12 myocytes (see FIG. 4a) enhancedbinding of both globular and full-length Adipo (see FIGS. 4c, 4 d),which was associated with increases in PPARα ligand activity (see FIG.4e) and fatty acid oxidation (see FIG. 4f) by globular Adipo andfull-length Adipo in C2C12 myocytes. Expression of AdipoR2 in C2C12myocytes (see FIG. 4b) also enhanced binding of both globular Adipo andfull length-Adipo (see FIGS. 4c, 4 d), which was associated withincreases in fatty acid oxidation (see FIG. 4f) by globular andfull-length Adipo in C2C12 myocytes. FIG. 4a shows mouse AdipoR1 mRNAlevels and FIG. 4b mouse AdipoR2 mRNA levels, with results shown using awhite bar for Mock, a black bar for mouse AdipoR1, and a dotted whitebar for mouse AdipoR2. FIGS. 4c and 4 d show the binding isotherm of[¹²⁵I] globular Adipo (gAd) or full-length Adipo (Ad) binding to C2C12myocytes transfected with mouse AdipoR1 or mouse AdipoR2, with a whitesquare indicating Mock, a black square mouse AdipoR1 and a black circlemouse AdipoR2. FIG. 4e show PPARα ligand activity in C2C12 myocytestransfected with mouse AdipoR1 or AdipoR2 when the myocytes wereinfected with adenovirus containing LacZ or DN-α2 AMPK and treated for 7hours with the indicated concentrations (μg/mL) of globular Adipo orfull-length Adipo, with white bars indicating results for Mock and blackbars for mouse AdipoR1. FIG. 4f shows in vitro fatty acid oxidation inthe aforementioned C2C12 myocytes, with white bars indicating resultsfor Mock, black bars for mouse AdipoR1 and dotted white bars for mouseAdipoR2. In all these figures, the bars show mean±s.e. (n=3-5), with “*”indicating P<0.05 and “**” P<0.01.

[0178] Expression of dominant negative AMP kinase did not affectglobular and full-length Adipo-induced and AdipoR1 expression-dependentincreases in PPARα ligand activity. These data strongly suggest thatboth AdipoR1 and AdipoR2 can mediate binding of globular and full-lengthAdipo and stimulate increases in PPARα ligand activity and fatty acidoxidation by globular and full-length Adipo.

[0179] (6) Effects of siRNA on Binding and Action of AdipoR in Myocytes

[0180] To determine whether endogenous AdipoR1 and R2 mediate thespecific binding and metabolic effects of Adipo in muscle cells, AdipoR1and R2 expression was suppressed using siRNA (see FIGS. 5a, 5 b). FIG.5a shows mouse AdipoR1 mRNA levels in C2C12 myocytes transfected withsiRNA or mock, while FIG. 5b shows mouse AdipoR2 mRNA levels in theC2C12 myocytes. In FIGS. 5a and 5 b, lane 1 shows results using mock,lane 2 using unrelated siRNA, lane 3 using siRNA for mouse AdipoR1 geneand lane 4 using siRNA for mouse AdipoR2 gene.

[0181] Suppression of AdipoR1 expression by siRNA (see FIG. 5a) in C2C12myocytes abolished globular Adipo binding activity and partially reducedfull-length Adipo binding activity (see FIGS. 5c, 5 d). FIG. 5c showsresults (n=4) of a competitive radioligand binding assay in which [¹²⁵I]globular Adipo binding to cells transfected with siRNA duplex wasreplaced by increasing the concentration of unlabeled globular Adipo.FIG. 5d shows results (n=4) of a competitive radioligand binding assayin which [¹²⁵I] full-length Adipo binding to cells transfected withsiRNA duplex was replaced by increasing the concentration of unlabeledfull-length Adipo.

[0182] Treatment with either globular or full-length Adipo for 7 hoursincreased PPARα ligand activity (see FIG. 5g) and stimulated fatty-acidoxidation (see FIG. 5h) and glucose uptake (see FIG. 5i) in C2C12myocytes. FIG. 5g shows PPARα ligand activity when C2C12 myocytestransfected with siRNA duplex were incubated for 7 hours with theindicated concentrations (μg/mL) of globular or full-length Adipo orwith 10⁻⁵ M Wy-14,643 (“Wy” in the figure). FIG. 5h shows in vitro fattyacid oxidation when C2C12 myocytes transfected with siRNA duplex wereincubated for 7 hours with the indicated concentrations (μg/mL) ofglobular or full-length Adipo or with 10⁻⁵ M Wy-14,643 (“Wy” in thefigure). FIG. 5i shows glucose uptake when C2C12 myocytes transfectedwith siRNA duplex were incubated for 7 hours with the indicatedconcentrations (μg/mL) of globular or full-length Adipo or with 10⁻⁷ Minsulin (“Ins” in the figure). In FIGS. 5g-5 i, “gAd0.01,” “gAd0.03,”“gAd0.1,” “gAd0.5” and “gAd2.5” indicate 0.01, 0.03, 0.1, 0.5 and 2.5μg/mL of globular Adipo, respectively, while “Ad0.1,” “Ad0.3,” “Ad1,”“Ad5” and “Ad25” indicate 0.1, 0.3, 1, 5 and 25 μg/mL of full-lengthAdipo, respectively. In the figures, each bar shows mean±s.e. (n=3-5),with “*” indicating P<0.05 and “**” P<0.01. In FIGS. 5g-5 i, white barsindicate results using unrelated siRNA, black bars using siRNA for mouseAdipoR1, dotted white bars using siRNA for mouse AdipoR2, and shadedbars using siRNA for mouse AdipoR1 and mouse AdipoR2.

[0183] Suppression of AdipoR1 expression by siRNA in C2C12 myocytes (seeFIG. 5a) reduced increases in PPARα ligand activity (see FIG. 5g), fattyacid oxidation (see FIG. 5h) and glucose uptake (see FIG. 5i) byglobular Adipo. In contrast, suppression of AdipoR1 expression failed tosignificantly reduce these effects by full-length Adipo. Thus, AdipoR1appears to mediate increases in PPARα ligand activity, fatty acidoxidation and glucose uptake by globular Adipo in muscle cells.

[0184] To determine whether endogenous AdipoR2 mediates the specificbinding and metabolic effects of Adipo in muscle cells, AdipoR2expression was suppressed using siRNA (see FIG. 5b). Suppression ofAdipoR2 expression by siRNA in C2C12 myocytes (see FIG. 5b) partiallyreduced both globular and full-length Adipo binding (see FIGS. 5e, 5 f).Moreover, suppression of AdipoR2 expression partially reduced increasesin PPARα ligand activity (see FIG. 5g) and fatty acid oxidation (seeFIG. 5h) by full-length Adipo. Thus, AdipoR2 appears to partiallymediate increases in PPARα ligand activity and fatty acid oxidation byfull-length Adipo in muscle cells. FIG. 5e shows the binding isotherm of[¹²⁵I] globular Adipo binding to C2C12 myocytes transfected with siRNAduplex, and FIG. 5f shows the binding isotherm of [¹²⁵I] full-lengthAdipo binding to C2C12 myocytes transfected with siRNA duplex. In thefigures, white squares indicate results using unrelated control siRNA,black circles using siRNA for mouse AdipoR2, and black triangles usingsiRNA for mouse AdipoR1 and mouse AdipoR2.

[0185] It appears that AdipoR1 is a receptor with relative selectivityfor globular Adipo, and AdipoR2 is a receptor with relative selectivityfor full-length Adipo. However, suppressing the functional expression ofeither type of AdipoR has significant effects on both globular andfull-length Adipo. These results may be explained by the observationthat AdipoR1 and AdipoR2 may form both homo- and hetero-multimers (seeFIG. 3e).

[0186] Interestingly, simultaneous suppression of AdipoR1 and AdipoR2expression with siRNA in C2C12 myocytes almost abolished both globularAdipo and full-length Adipo binding (see FIG. 5e, 5 f), and increases inPPARα ligand activity (see FIG. 5g) and fatty acid oxidation (see FIG.5h) by globular and full-length Adipo.

[0187] (7) Effects of siRNA on Binding and Action in Hepatocytes

[0188] The binding of Adipo to hepatocytes was studied. Hepatocytesshowed specific binding for full-length Adipo (see FIG. 6b). Expressionof AdipoR1 and R2 enhanced binding of globular and full-length Adipo tohepatocytes (see FIGS. 6a, 6 b). Conversely, suppression of AdipoR2expression by siRNA in hepatocytes greatly reduced full-length Adipobinding (see FIG. 6b). These data appear to suggest that AdipoR1 is areceptor with relative selectivity for globular Adipo, while AdipoR2 isa receptor with relative selectivity for full-length Adipo.

[0189] The binding of Adipo to Normal Human Aortic Endothelial Cells(HAEC) was studied. Simultaneous suppression of both AdipoR1 and AdipoR2expression with siRNA greatly reduced globular Adipo binding (see FIG.6e), and partially reduced full-length Adipo binding (see FIG. 6f).These data indicate that AdipoR1 and AdipoR2 are also receptors forAdipo in HAEC.

[0190] (8) Scatchard Plot Analysis

[0191]FIGS. 7a and 7 b show the results of the tests of FIGS. 5e and 5 fconducted again in more detail. FIG. 7a (which corresponds to FIG. 5e)shows the binding isotherm of [¹²⁵I] globular Adipo binding to C2C12myocytes transfected with siRNA duplex, while FIG. 7b (which correspondsto FIG. 5f) shows the binding isotherm of [¹²⁵I] full-length Adipobinding to C2C12 myocytes transfected with siRNA duplex. In FIGS. 7a and7 b, white squares indicate results using unrelated control siRNA, blacksquares using siRNA for mouse AdipoR1, black circles using siRNA formouse AdipoR2, and black triangles using siRNA for mouse AdipoR1 andmouse AdipoR2.

[0192] Scatchard plot analysis was carried out based on the resultsshown in FIGS. 7a and 7 b, with the results shown in FIGS. 7c and 7 d.

[0193] C2C12 myocytes transfected with unrelated siRNA bound morestrongly to globular Adipo than to full-length Adipo (see FIGS. 7a and 7b).

[0194] Scatchard plot analysis revealed that there are two types ofbinding sites for globular Adipo; high affinity binding sites (Kd valueabout 0.06 μg/mL, equivalent to 1.14 nM of gAd trimer) and intermediateaffinity binding sites (Kd value about 0.80 μg/mL, equivalent to 14.4 nMof gAd trimer) (see FIG. 7c), and there are also two types of bindingsites for full-length Adipo; intermediate affinity binding sites (Kdvalue about 6.7 μg/mL, equivalent to 49.1 nM of Ad hexamer) and lowaffinity binding sites (Kd value about 329.3 μg/mL, equivalent to 2415nM of Ad hexamer) (see FIG. 7d).

[0195] Suppression of AdipoR1 expression with siRNA largely reducedglobular Adipo binding (see FIG. 7a), but only barely reducedfull-length Adipo binding (see FIG. 7b). Scatchard plot analysisrevealed that specific suppression of AdipoR1 abrogated high affinitybinding sites for globular Adipo, but failed to affect intermediateaffinity binding sites for globular Adipo (see FIG. 7c). Moreover,Scatchard plot analysis revealed that specific suppression of AdipoR1expression only partially reduced the activity of intermediate affinitybinding sites for full-length Adipo, but abrogated low affinity bindingsites for full-length Adipo (see FIG. 7d).

[0196] By contrast with AdipoR1, suppression of AdipoR2 expression withsiRNA largely reduced full-length Adipo binding (see FIG. 7b), butbarely reduced globular Adipo binding (see FIG. 7a). Scatchard plotanalysis revealed that specific suppression of AdipoR2 only partlyreduced the activity of high affinity binding sites for globular Adipo,but abrogated intermediate affinity binding sites for globular Adipo(see FIG. 7c). Moreover, Scatchard plot analysis revealed that specificsuppression of AdipoR2 abrogated intermediate affinity binding sites forfull-length Adipo, but failed to affect the activity of low affinitybinding sites for full-length Adipo (see FIG. 7d).

[0197] (9) Phosphorylation and Amount of Phosphorylation of AMPK, ACC,p38 MAPK and MAPK

[0198] The panels in FIG. 8a show the results when C2C12 cellstransfected (“AdipoR1” in figures) or not transfected (“Mock” infigures) with AdipoR1 were incubated for 10 minutes with 0.1 or 1 μg/mLof gAd, and a lysate of those cells was then reacted withanti-phosphorylated AMPK antibodies, while the panels in FIG. 8b showthe results when a lysate of those cells was reacted withanti-phosphorylated ACC antibodies, the panels in FIG. 8c show theresults when a lysate of those cells was reacted withanti-phosphorylated p38 MAPK antibodies, and the panels in FIG. 8d showthe results when a lysate of those cells was reacted withanti-phosphorylated MAPK antibodies. The panels in FIG. 8e show theresults when hepatocytes transfected (“AdipoR1” in figures) or nottransfected (“Mock” in figures) with AdipoR1 were incubated for 10minutes with 0.1 or 1 μg/mL of gAd and a lysate of those cells was thenreacted with anti-phosphorylated AMPK antibodies, while the panels inFIG. 8f show the results when a lysate of those cells was reacted withanti-phosphorylated ACC antibodies. The graphs below the panels in FIGS.8a-8 f show amounts of phosphorylation at each position on the panel. Inthe figures, “pAMPK” is phosphorylated AMPK, “PACC” is phosphorylatedACC, “pp38 MAPK” is phosphorylated p38 MAPK, “pp44 MAPK isphosphorylated p44MAPK, and “pp42 MAPK” is phosphorylated p42 MAPK.

[0199] In C2C12 myocytes not transfected with AdipoR1, both globularAdipo and full-length Adipo increased amounts of phosphorylation ofAMPK, ACC and p38 MAPK, but did not increase phosphorylation of MAPK andother protein kinases (see FIGS. 8a-8 d). Expression of AdipoR1 in C2C12cells was related to stimulation of phosphorylation of AMPK, ACC and p38MAPK by globular Adipo (see FIGS. 8a-8 d). This suggests that AdipoR1mediates activation of AMPK and p38 MAPK by globular Adipo.

[0200] In hepatocytes not transfected with AdipoR1, full-length Adipopromoted AMPK activation and ACC phosphorylation, while globular Adipodid not (see FIGS. 8e and 8 f). Expression of AdipoR1 in hepatocytes wasrelated to stimulation of AMPK and ACC phosphorylation by globular andfull-length Adipo (see FIGS. 8e and 8 f). This suggests that AdipoR1mediates AMPK and ACC phosphorylation by globular and full-length Adipo.

[0201] In C2C12 myocytes not transfected with AdipoR1 (“Mock” in FIG.8), the fatty acid oxidation and glucose uptake stimulated by globularAdipo were partially inhibited by dominant negative (DN) AMPK or the p38MAPK specific inhibitor SB203580 (Barger, P. M. et al, J. Biol. Chem.276, 44495-44501 (2001); Puigserver, P. et al, Mol. Cell 8, 971-982(2001); Michael, L. F. et al, Proc. Natl. Acad. Sci. USA 98, 3820-3825(2001)) (see FIGS. 8g and 8 h). Expression of AdipoR1 in C2C12 myocytes(“mAdipoR1” in FIG. 8) promoted fatty acid oxidation and glucose uptakeby globular Adipo, but this effect was also partially inhibited byDN-AMPK or SB203580 (see FIGS. 8g and 8 h). Thus, the stimulation offatty acid oxidation and glucose uptake by globular Adipo via AdipoR1appeared to be associated with both AMPK and p38 MAPK pathways in C2C12myocytes.

[0202] From the results above, it appears that the AdipoR1 cDNA (Seq.Nos. 1 & 5) and AdipoR2 cDNA (Seq. Nos. 3 & 7) obtained in theseexamples encodes AdipoR1 (Seq. Nos. 2 & 6) and AdipoR2 (Seq. Nos. 4 & 8)having biological functions.

INDUSTRIAL APPLICABILITY

[0203] The present invention provides a novel protein having adiponectinbinding ability, a gene encoding the aforementioned protein, arecombinant vector containing the aforementioned gene, a transformantcontaining the aforementioned recombinant vector and and antibody to theproteins. Moreover, the present invention provides a screening methodand screening kit for screening a ligand, agonist and antagonist to anadiponectin receptor using the aforementioned protein, gene, recombinantvector or transformant.

1 8 1 1128 DNA Homo sapiens CDS (1)...(1125) 1 atg tct tcc cac aaa ggatct gtg gtg gca cag ggg aat ggg gct cct 48 Met Ser Ser His Lys Gly SerVal Val Ala Gln Gly Asn Gly Ala Pro 1 5 10 15 gcc agt aac agg gaa gctgac acg gtg gaa ctg gct gaa ctg gga ccc 96 Ala Ser Asn Arg Glu Ala AspThr Val Glu Leu Ala Glu Leu Gly Pro 20 25 30 ctg cta gaa gag aag ggc aaacgg gta atc gcc aac cca ccc aaa gct 144 Leu Leu Glu Glu Lys Gly Lys ArgVal Ile Ala Asn Pro Pro Lys Ala 35 40 45 gaa gaa gag caa aca tgc cca gtgccc cag gaa gaa gag gag gag gtg 192 Glu Glu Glu Gln Thr Cys Pro Val ProGln Glu Glu Glu Glu Glu Val 50 55 60 cgg gta ctg aca ctt ccc ctg caa gcccac cac gcc atg gag aag atg 240 Arg Val Leu Thr Leu Pro Leu Gln Ala HisHis Ala Met Glu Lys Met 65 70 75 80 gaa gag ttt gtg tac aag gtc tgg gaggga cgt tgg agg gtc atc cca 288 Glu Glu Phe Val Tyr Lys Val Trp Glu GlyArg Trp Arg Val Ile Pro 85 90 95 tat gat gtg ctc cct gac tgg cta aag gacaac gac tat ctg cta cat 336 Tyr Asp Val Leu Pro Asp Trp Leu Lys Asp AsnAsp Tyr Leu Leu His 100 105 110 ggt cat aga cct ccc atg ccc tcc ttt cgggct tgc ttc aag agc atc 384 Gly His Arg Pro Pro Met Pro Ser Phe Arg AlaCys Phe Lys Ser Ile 115 120 125 ttc cgc att cat aca gaa act ggc aac atctgg acc cat ctg ctt ggt 432 Phe Arg Ile His Thr Glu Thr Gly Asn Ile TrpThr His Leu Leu Gly 130 135 140 ttc gtg ctg ttt ctc ttt ttg gga atc ttgacc atg ctc aga cca aat 480 Phe Val Leu Phe Leu Phe Leu Gly Ile Leu ThrMet Leu Arg Pro Asn 145 150 155 160 atg tac ttc atg gcc cct cta cag gagaag gtg gtt ttt ggg atg ttc 528 Met Tyr Phe Met Ala Pro Leu Gln Glu LysVal Val Phe Gly Met Phe 165 170 175 ttt ttg ggt gca gtg ctc tgc ctc agcttc tcc tgg ctc ttt cac acc 576 Phe Leu Gly Ala Val Leu Cys Leu Ser PheSer Trp Leu Phe His Thr 180 185 190 gtc tat tgt cat tca gag aaa gtc tctcgg act ttt tcc aaa ctg gac 624 Val Tyr Cys His Ser Glu Lys Val Ser ArgThr Phe Ser Lys Leu Asp 195 200 205 tat tca ggg att gct ctt cta att atgggg agc ttt gtc ccc tgg ctc 672 Tyr Ser Gly Ile Ala Leu Leu Ile Met GlySer Phe Val Pro Trp Leu 210 215 220 tat tat tcc ttc tac tgc tcc cca cagcca cgg ctc atc tac ctc tcc 720 Tyr Tyr Ser Phe Tyr Cys Ser Pro Gln ProArg Leu Ile Tyr Leu Ser 225 230 235 240 atc gtc tgt gtc ctg ggc att tctgcc atc att gtg gcg cag tgg gac 768 Ile Val Cys Val Leu Gly Ile Ser AlaIle Ile Val Ala Gln Trp Asp 245 250 255 cgg ttt gcc act cct aag cac cggcag aca aga gca ggc gtg ttc ctg 816 Arg Phe Ala Thr Pro Lys His Arg GlnThr Arg Ala Gly Val Phe Leu 260 265 270 gga ctt ggc ttg agt ggc gtc gtgccc acc atg cac ttt act atc gct 864 Gly Leu Gly Leu Ser Gly Val Val ProThr Met His Phe Thr Ile Ala 275 280 285 gag ggc ttt gtc aag gcc acc acagtg ggc cag atg ggc tgg ttc ttc 912 Glu Gly Phe Val Lys Ala Thr Thr ValGly Gln Met Gly Trp Phe Phe 290 295 300 ctc atg gct gtg atg tac atc actgga gct ggc ctt tat gct gct cga 960 Leu Met Ala Val Met Tyr Ile Thr GlyAla Gly Leu Tyr Ala Ala Arg 305 310 315 320 att cct gag cgc ttc ttt cctgga aaa ttt gac ata tgg ttc cag tct 1008 Ile Pro Glu Arg Phe Phe Pro GlyLys Phe Asp Ile Trp Phe Gln Ser 325 330 335 cat cag att ttc cat gtc ctggtg gtg gca gca gcc ttt gtc cac ttc 1056 His Gln Ile Phe His Val Leu ValVal Ala Ala Ala Phe Val His Phe 340 345 350 tat gga gtc tcc aac ctt caggaa ttc cgt tac ggc cta gaa ggc ggc 1104 Tyr Gly Val Ser Asn Leu Gln GluPhe Arg Tyr Gly Leu Glu Gly Gly 355 360 365 tgt act gat gac acc ctt ctctga 1128 Cys Thr Asp Asp Thr Leu Leu 370 375 2 375 PRT Homo sapiens 2Met Ser Ser His Lys Gly Ser Val Val Ala Gln Gly Asn Gly Ala Pro 1 5 1015 Ala Ser Asn Arg Glu Ala Asp Thr Val Glu Leu Ala Glu Leu Gly Pro 20 2530 Leu Leu Glu Glu Lys Gly Lys Arg Val Ile Ala Asn Pro Pro Lys Ala 35 4045 Glu Glu Glu Gln Thr Cys Pro Val Pro Gln Glu Glu Glu Glu Glu Val 50 5560 Arg Val Leu Thr Leu Pro Leu Gln Ala His His Ala Met Glu Lys Met 65 7075 80 Glu Glu Phe Val Tyr Lys Val Trp Glu Gly Arg Trp Arg Val Ile Pro 8590 95 Tyr Asp Val Leu Pro Asp Trp Leu Lys Asp Asn Asp Tyr Leu Leu His100 105 110 Gly His Arg Pro Pro Met Pro Ser Phe Arg Ala Cys Phe Lys SerIle 115 120 125 Phe Arg Ile His Thr Glu Thr Gly Asn Ile Trp Thr His LeuLeu Gly 130 135 140 Phe Val Leu Phe Leu Phe Leu Gly Ile Leu Thr Met LeuArg Pro Asn 145 150 155 160 Met Tyr Phe Met Ala Pro Leu Gln Glu Lys ValVal Phe Gly Met Phe 165 170 175 Phe Leu Gly Ala Val Leu Cys Leu Ser PheSer Trp Leu Phe His Thr 180 185 190 Val Tyr Cys His Ser Glu Lys Val SerArg Thr Phe Ser Lys Leu Asp 195 200 205 Tyr Ser Gly Ile Ala Leu Leu IleMet Gly Ser Phe Val Pro Trp Leu 210 215 220 Tyr Tyr Ser Phe Tyr Cys SerPro Gln Pro Arg Leu Ile Tyr Leu Ser 225 230 235 240 Ile Val Cys Val LeuGly Ile Ser Ala Ile Ile Val Ala Gln Trp Asp 245 250 255 Arg Phe Ala ThrPro Lys His Arg Gln Thr Arg Ala Gly Val Phe Leu 260 265 270 Gly Leu GlyLeu Ser Gly Val Val Pro Thr Met His Phe Thr Ile Ala 275 280 285 Glu GlyPhe Val Lys Ala Thr Thr Val Gly Gln Met Gly Trp Phe Phe 290 295 300 LeuMet Ala Val Met Tyr Ile Thr Gly Ala Gly Leu Tyr Ala Ala Arg 305 310 315320 Ile Pro Glu Arg Phe Phe Pro Gly Lys Phe Asp Ile Trp Phe Gln Ser 325330 335 His Gln Ile Phe His Val Leu Val Val Ala Ala Ala Phe Val His Phe340 345 350 Tyr Gly Val Ser Asn Leu Gln Glu Phe Arg Tyr Gly Leu Glu GlyGly 355 360 365 Cys Thr Asp Asp Thr Leu Leu 370 375 3 900 DNA Homosapiens CDS (1)...(897) 3 atg gaa aaa atg gaa gaa ttt gtt tgt aag gtatgg gaa ggt cgg tgg 48 Met Glu Lys Met Glu Glu Phe Val Cys Lys Val TrpGlu Gly Arg Trp 1 5 10 15 cga gtg atc cct cat gat gta cta cca gac tggctc aag gat aat gac 96 Arg Val Ile Pro His Asp Val Leu Pro Asp Trp LeuLys Asp Asn Asp 20 25 30 ttc ctc ttg cat gga cac cgg cct cct atg cct tctttc cgg gcc tgt 144 Phe Leu Leu His Gly His Arg Pro Pro Met Pro Ser PheArg Ala Cys 35 40 45 ttt aag agc att ttc aga ata cac aca gaa aca ggc aacatt tgg aca 192 Phe Lys Ser Ile Phe Arg Ile His Thr Glu Thr Gly Asn IleTrp Thr 50 55 60 cat ctc tta ggt tgt gta ttc ttc ctg tgc ctg ggg atc ttttat atg 240 His Leu Leu Gly Cys Val Phe Phe Leu Cys Leu Gly Ile Phe TyrMet 65 70 75 80 ttt cgc cca aat atc tcc ttt gtg gcc cct ctg caa gag aaggtg gtc 288 Phe Arg Pro Asn Ile Ser Phe Val Ala Pro Leu Gln Glu Lys ValVal 85 90 95 ttt gga tta ttt ttc tta gga gcc att ctc tgc ctt tct ttt tcatgg 336 Phe Gly Leu Phe Phe Leu Gly Ala Ile Leu Cys Leu Ser Phe Ser Trp100 105 110 ctc ttc cac aca gtc tac tgc cac tca gag ggg gtc tct cgg ctcttc 384 Leu Phe His Thr Val Tyr Cys His Ser Glu Gly Val Ser Arg Leu Phe115 120 125 tct aaa ctg gat tac tct ggt att gct ctt ctg att atg gga agtttt 432 Ser Lys Leu Asp Tyr Ser Gly Ile Ala Leu Leu Ile Met Gly Ser Phe130 135 140 gtt cct tgg ctt tat tat tct ttc tac tgt aat cca caa cct tgcttc 480 Val Pro Trp Leu Tyr Tyr Ser Phe Tyr Cys Asn Pro Gln Pro Cys Phe145 150 155 160 atc tac ttg att gtc atc tgt gtg ctg ggc att gca gcc attata gtc 528 Ile Tyr Leu Ile Val Ile Cys Val Leu Gly Ile Ala Ala Ile IleVal 165 170 175 tcc cag tgg gac atg ttt gcc acc cct cag tat cgg gga gtaaga gca 576 Ser Gln Trp Asp Met Phe Ala Thr Pro Gln Tyr Arg Gly Val ArgAla 180 185 190 gga gtg ttt ttg ggc cta ggc ctg agt gga atc att cct accttg cac 624 Gly Val Phe Leu Gly Leu Gly Leu Ser Gly Ile Ile Pro Thr LeuHis 195 200 205 tat gtc atc tcg gag ggg ttc ctt aag gcc gcc acc ata gggcag ata 672 Tyr Val Ile Ser Glu Gly Phe Leu Lys Ala Ala Thr Ile Gly GlnIle 210 215 220 ggc tgg ttg atg ctg atg gcc agc ctc tac atc aca gga gctgcc ctg 720 Gly Trp Leu Met Leu Met Ala Ser Leu Tyr Ile Thr Gly Ala AlaLeu 225 230 235 240 tat gct gcc cgg atc ccc gaa cgc ttt ttc cct ggc aaatgt gac atc 768 Tyr Ala Ala Arg Ile Pro Glu Arg Phe Phe Pro Gly Lys CysAsp Ile 245 250 255 tgg ttt cac tct cat cag ctg ttt cat atc ttt gtg gttgct gga gct 816 Trp Phe His Ser His Gln Leu Phe His Ile Phe Val Val AlaGly Ala 260 265 270 ttt gtt cac ttc cat ggt gtc tca aac ctc cag gag tttcgt ttc atg 864 Phe Val His Phe His Gly Val Ser Asn Leu Gln Glu Phe ArgPhe Met 275 280 285 atc ggc ggg ggc tgc agt gaa gag gat gca ctg tga 900Ile Gly Gly Gly Cys Ser Glu Glu Asp Ala Leu 290 295 4 299 PRT Homosapiens 4 Met Glu Lys Met Glu Glu Phe Val Cys Lys Val Trp Glu Gly ArgTrp 1 5 10 15 Arg Val Ile Pro His Asp Val Leu Pro Asp Trp Leu Lys AspAsn Asp 20 25 30 Phe Leu Leu His Gly His Arg Pro Pro Met Pro Ser Phe ArgAla Cys 35 40 45 Phe Lys Ser Ile Phe Arg Ile His Thr Glu Thr Gly Asn IleTrp Thr 50 55 60 His Leu Leu Gly Cys Val Phe Phe Leu Cys Leu Gly Ile PheTyr Met 65 70 75 80 Phe Arg Pro Asn Ile Ser Phe Val Ala Pro Leu Gln GluLys Val Val 85 90 95 Phe Gly Leu Phe Phe Leu Gly Ala Ile Leu Cys Leu SerPhe Ser Trp 100 105 110 Leu Phe His Thr Val Tyr Cys His Ser Glu Gly ValSer Arg Leu Phe 115 120 125 Ser Lys Leu Asp Tyr Ser Gly Ile Ala Leu LeuIle Met Gly Ser Phe 130 135 140 Val Pro Trp Leu Tyr Tyr Ser Phe Tyr CysAsn Pro Gln Pro Cys Phe 145 150 155 160 Ile Tyr Leu Ile Val Ile Cys ValLeu Gly Ile Ala Ala Ile Ile Val 165 170 175 Ser Gln Trp Asp Met Phe AlaThr Pro Gln Tyr Arg Gly Val Arg Ala 180 185 190 Gly Val Phe Leu Gly LeuGly Leu Ser Gly Ile Ile Pro Thr Leu His 195 200 205 Tyr Val Ile Ser GluGly Phe Leu Lys Ala Ala Thr Ile Gly Gln Ile 210 215 220 Gly Trp Leu MetLeu Met Ala Ser Leu Tyr Ile Thr Gly Ala Ala Leu 225 230 235 240 Tyr AlaAla Arg Ile Pro Glu Arg Phe Phe Pro Gly Lys Cys Asp Ile 245 250 255 TrpPhe His Ser His Gln Leu Phe His Ile Phe Val Val Ala Gly Ala 260 265 270Phe Val His Phe His Gly Val Ser Asn Leu Gln Glu Phe Arg Phe Met 275 280285 Ile Gly Gly Gly Cys Ser Glu Glu Asp Ala Leu 290 295 5 1128 DNA Musmusculus CDS (1)...(1125) 5 atg tct tcc cac aaa ggc tct gcc ggg gca caaggc aat ggg gct cct 48 Met Ser Ser His Lys Gly Ser Ala Gly Ala Gln GlyAsn Gly Ala Pro 1 5 10 15 tct ggt aac aga gaa gct gac aca gtg gag ctggct gag ctg ggg ccc 96 Ser Gly Asn Arg Glu Ala Asp Thr Val Glu Leu AlaGlu Leu Gly Pro 20 25 30 ctg ctg gag gag aag ggc aag cgg gca gcc agc agccca gcc aag gct 144 Leu Leu Glu Glu Lys Gly Lys Arg Ala Ala Ser Ser ProAla Lys Ala 35 40 45 gag gaa gat caa gca tgc ccg gtg cct cag gaa gag gaggag gag gtg 192 Glu Glu Asp Gln Ala Cys Pro Val Pro Gln Glu Glu Glu GluGlu Val 50 55 60 cgg gtg ctg acg ctt cct ctg caa gcc cac cat gcc atg gagaag atg 240 Arg Val Leu Thr Leu Pro Leu Gln Ala His His Ala Met Glu LysMet 65 70 75 80 gag gag ttc gtg tat aag gtc tgg gag gga cgt tgg aga gtcatc ccg 288 Glu Glu Phe Val Tyr Lys Val Trp Glu Gly Arg Trp Arg Val IlePro 85 90 95 tat gat gtg ctt cct gac tgg ctg aaa gac aac gac tac ctg ctacat 336 Tyr Asp Val Leu Pro Asp Trp Leu Lys Asp Asn Asp Tyr Leu Leu His100 105 110 ggc cac aga cca cct atg ccc tcc ttt cgg gct tgc ttc aag agcatc 384 Gly His Arg Pro Pro Met Pro Ser Phe Arg Ala Cys Phe Lys Ser Ile115 120 125 ttc cgc atc cac aca gag act ggc aac atc tgg aca cat ctg cttggt 432 Phe Arg Ile His Thr Glu Thr Gly Asn Ile Trp Thr His Leu Leu Gly130 135 140 ttt gtg cta ttt ctc ttt ctg gga atc ttg acg atg ctg aga ccaaat 480 Phe Val Leu Phe Leu Phe Leu Gly Ile Leu Thr Met Leu Arg Pro Asn145 150 155 160 atg tac ttc atg gct ccc ctg cag gag aag gtg gtc ttc gggatg ttc 528 Met Tyr Phe Met Ala Pro Leu Gln Glu Lys Val Val Phe Gly MetPhe 165 170 175 ttc ctg ggc gcg gtg ctc tgc ctc agt ttc tcc tgg ctc ttccac act 576 Phe Leu Gly Ala Val Leu Cys Leu Ser Phe Ser Trp Leu Phe HisThr 180 185 190 gtc tac tgt cat tca gag aag gtc tct cgg act ttt tcc aaactg gac 624 Val Tyr Cys His Ser Glu Lys Val Ser Arg Thr Phe Ser Lys LeuAsp 195 200 205 tat tca ggg att gct cta ctg att atg ggg agc ttc gtt ccctgg ctc 672 Tyr Ser Gly Ile Ala Leu Leu Ile Met Gly Ser Phe Val Pro TrpLeu 210 215 220 tat tac tcc ttc tac tgc tcc cca cag ccg cgg ctc atc tacctc tcc 720 Tyr Tyr Ser Phe Tyr Cys Ser Pro Gln Pro Arg Leu Ile Tyr LeuSer 225 230 235 240 atc gtc tgt gtc ctg ggc atc tct gcc atc att gtg gcacag tgg gac 768 Ile Val Cys Val Leu Gly Ile Ser Ala Ile Ile Val Ala GlnTrp Asp 245 250 255 cgg ttt gcc act ccc aag cac cgg cag aca aga gca ggagtg ttc ctg 816 Arg Phe Ala Thr Pro Lys His Arg Gln Thr Arg Ala Gly ValPhe Leu 260 265 270 gga ctt ggc ttg agt ggt gtt gta ccc acc atg cac tttact atc gct 864 Gly Leu Gly Leu Ser Gly Val Val Pro Thr Met His Phe ThrIle Ala 275 280 285 gag ggc ttt gtc aag gcc acc acg gtg ggc cag atg ggctgg ttc ttc 912 Glu Gly Phe Val Lys Ala Thr Thr Val Gly Gln Met Gly TrpPhe Phe 290 295 300 ctc atg gct gtg atg tac atc acc ggc gcc ggc ctg tatgct gct cgg 960 Leu Met Ala Val Met Tyr Ile Thr Gly Ala Gly Leu Tyr AlaAla Arg 305 310 315 320 att cct gag cgc ttc ttc cct gga aaa ttt gac atctgg ttc cag tct 1008 Ile Pro Glu Arg Phe Phe Pro Gly Lys Phe Asp Ile TrpPhe Gln Ser 325 330 335 cat cag att ttc cac gtc ctg gtg gtg gca gca gctttc gtc cac ttc 1056 His Gln Ile Phe His Val Leu Val Val Ala Ala Ala PheVal His Phe 340 345 350 tat ggt gtg tcc aac ctt cag gaa ttc cgt tat ggccta gaa ggt ggc 1104 Tyr Gly Val Ser Asn Leu Gln Glu Phe Arg Tyr Gly LeuGlu Gly Gly 355 360 365 tgt acc gac gac tcc ctt ctc tga 1128 Cys Thr AspAsp Ser Leu Leu 370 375 6 375 PRT Mus musculus 6 Met Ser Ser His Lys GlySer Ala Gly Ala Gln Gly Asn Gly Ala Pro 1 5 10 15 Ser Gly Asn Arg GluAla Asp Thr Val Glu Leu Ala Glu Leu Gly Pro 20 25 30 Leu Leu Glu Glu LysGly Lys Arg Ala Ala Ser Ser Pro Ala Lys Ala 35 40 45 Glu Glu Asp Gln AlaCys Pro Val Pro Gln Glu Glu Glu Glu Glu Val 50 55 60 Arg Val Leu Thr LeuPro Leu Gln Ala His His Ala Met Glu Lys Met 65 70 75 80 Glu Glu Phe ValTyr Lys Val Trp Glu Gly Arg Trp Arg Val Ile Pro 85 90 95 Tyr Asp Val LeuPro Asp Trp Leu Lys Asp Asn Asp Tyr Leu Leu His 100 105 110 Gly His ArgPro Pro Met Pro Ser Phe Arg Ala Cys Phe Lys Ser Ile 115 120 125 Phe ArgIle His Thr Glu Thr Gly Asn Ile Trp Thr His Leu Leu Gly 130 135 140 PheVal Leu Phe Leu Phe Leu Gly Ile Leu Thr Met Leu Arg Pro Asn 145 150 155160 Met Tyr Phe Met Ala Pro Leu Gln Glu Lys Val Val Phe Gly Met Phe 165170 175 Phe Leu Gly Ala Val Leu Cys Leu Ser Phe Ser Trp Leu Phe His Thr180 185 190 Val Tyr Cys His Ser Glu Lys Val Ser Arg Thr Phe Ser Lys LeuAsp 195 200 205 Tyr Ser Gly Ile Ala Leu Leu Ile Met Gly Ser Phe Val ProTrp Leu 210 215 220 Tyr Tyr Ser Phe Tyr Cys Ser Pro Gln Pro Arg Leu IleTyr Leu Ser 225 230 235 240 Ile Val Cys Val Leu Gly Ile Ser Ala Ile IleVal Ala Gln Trp Asp 245 250 255 Arg Phe Ala Thr Pro Lys His Arg Gln ThrArg Ala Gly Val Phe Leu 260 265 270 Gly Leu Gly Leu Ser Gly Val Val ProThr Met His Phe Thr Ile Ala 275 280 285 Glu Gly Phe Val Lys Ala Thr ThrVal Gly Gln Met Gly Trp Phe Phe 290 295 300 Leu Met Ala Val Met Tyr IleThr Gly Ala Gly Leu Tyr Ala Ala Arg 305 310 315 320 Ile Pro Glu Arg PhePhe Pro Gly Lys Phe Asp Ile Trp Phe Gln Ser 325 330 335 His Gln Ile PheHis Val Leu Val Val Ala Ala Ala Phe Val His Phe 340 345 350 Tyr Gly ValSer Asn Leu Gln Glu Phe Arg Tyr Gly Leu Glu Gly Gly 355 360 365 Cys ThrAsp Asp Ser Leu Leu 370 375 7 936 DNA Mus musculus CDS (1)...(933) 7 atgggc atg tcc ccg ctc cta cag gcc cat cat gct atg gaa cga atg 48 Met GlyMet Ser Pro Leu Leu Gln Ala His His Ala Met Glu Arg Met 1 5 10 15 gaagag ttt gtt tgt aag gtg tgg gaa ggc cga tgg cga gtg atc cct 96 Glu GluPhe Val Cys Lys Val Trp Glu Gly Arg Trp Arg Val Ile Pro 20 25 30 cac gatgtg cta ccg gat tgg ctt aag gat aat gac ttc ctt ctc cat 144 His Asp ValLeu Pro Asp Trp Leu Lys Asp Asn Asp Phe Leu Leu His 35 40 45 gga cac cggcct cct atg cct tcc ttt cgg gcc tgt ttt aag agc att 192 Gly His Arg ProPro Met Pro Ser Phe Arg Ala Cys Phe Lys Ser Ile 50 55 60 ttt aga ata cacaca gag acg ggc aac att tgg aca cat ctc cta ggt 240 Phe Arg Ile His ThrGlu Thr Gly Asn Ile Trp Thr His Leu Leu Gly 65 70 75 80 tgt gta ttc ttcctg tgc ctg ggg atc ttt tat atg ttt cgc cca aat 288 Cys Val Phe Phe LeuCys Leu Gly Ile Phe Tyr Met Phe Arg Pro Asn 85 90 95 ata tct ttt gtg gcccct ctg caa gag aaa gtg gtc ttt ggc ttg ttc 336 Ile Ser Phe Val Ala ProLeu Gln Glu Lys Val Val Phe Gly Leu Phe 100 105 110 ttc ttg gga gcc attctc tgc ctt tcc ttt tca tgg ctc ttc cac acg 384 Phe Leu Gly Ala Ile LeuCys Leu Ser Phe Ser Trp Leu Phe His Thr 115 120 125 gtg tac tgc cac tcagaa ggg gtc tcc cga ctc ttc tct aaa ttg gat 432 Val Tyr Cys His Ser GluGly Val Ser Arg Leu Phe Ser Lys Leu Asp 130 135 140 tac tct ggt att gctctt ctg atc atg gga agt ttt gtt cct tgg ctt 480 Tyr Ser Gly Ile Ala LeuLeu Ile Met Gly Ser Phe Val Pro Trp Leu 145 150 155 160 tat tat tct ttctac tgt aac cca caa cct tgc ttc atc tac ctg att 528 Tyr Tyr Ser Phe TyrCys Asn Pro Gln Pro Cys Phe Ile Tyr Leu Ile 165 170 175 gtc atc tgt gtgctg ggc att gca gcc att atc gtc tct cag tgg gac 576 Val Ile Cys Val LeuGly Ile Ala Ala Ile Ile Val Ser Gln Trp Asp 180 185 190 atg ttt gcc acccct cag tat cgg ggg gtc aga gca gga gtg ttc gtg 624 Met Phe Ala Thr ProGln Tyr Arg Gly Val Arg Ala Gly Val Phe Val 195 200 205 ggc tta ggc ctgagt gga atc atc cct acc ttg cat tat gtc atc tca 672 Gly Leu Gly Leu SerGly Ile Ile Pro Thr Leu His Tyr Val Ile Ser 210 215 220 gaa ggg ttc ctgaag gct gcc acc ata ggg cag ata ggc tgg cta atg 720 Glu Gly Phe Leu LysAla Ala Thr Ile Gly Gln Ile Gly Trp Leu Met 225 230 235 240 ctt atg gctagc ctc tat atc acc gga gct gcc ctc tat gcg gcc cgt 768 Leu Met Ala SerLeu Tyr Ile Thr Gly Ala Ala Leu Tyr Ala Ala Arg 245 250 255 atc cct gagcgc ttc ttt cct ggc aaa tgt gac atc tgg ttt cac tct 816 Ile Pro Glu ArgPhe Phe Pro Gly Lys Cys Asp Ile Trp Phe His Ser 260 265 270 cat cag ctcttc cac atc ttt gtg gtt gct ggt gcc ttt gtt cac ttc 864 His Gln Leu PheHis Ile Phe Val Val Ala Gly Ala Phe Val His Phe 275 280 285 cac gga gtctca aac ctg cag gaa ttt cgt ttc atg att ggc ggg ggc 912 His Gly Val SerAsn Leu Gln Glu Phe Arg Phe Met Ile Gly Gly Gly 290 295 300 tgc act gaagag gat gca ctg tga 936 Cys Thr Glu Glu Asp Ala Leu 305 310 8 311 PRTMus musculus 8 Met Gly Met Ser Pro Leu Leu Gln Ala His His Ala Met GluArg Met 1 5 10 15 Glu Glu Phe Val Cys Lys Val Trp Glu Gly Arg Trp ArgVal Ile Pro 20 25 30 His Asp Val Leu Pro Asp Trp Leu Lys Asp Asn Asp PheLeu Leu His 35 40 45 Gly His Arg Pro Pro Met Pro Ser Phe Arg Ala Cys PheLys Ser Ile 50 55 60 Phe Arg Ile His Thr Glu Thr Gly Asn Ile Trp Thr HisLeu Leu Gly 65 70 75 80 Cys Val Phe Phe Leu Cys Leu Gly Ile Phe Tyr MetPhe Arg Pro Asn 85 90 95 Ile Ser Phe Val Ala Pro Leu Gln Glu Lys Val ValPhe Gly Leu Phe 100 105 110 Phe Leu Gly Ala Ile Leu Cys Leu Ser Phe SerTrp Leu Phe His Thr 115 120 125 Val Tyr Cys His Ser Glu Gly Val Ser ArgLeu Phe Ser Lys Leu Asp 130 135 140 Tyr Ser Gly Ile Ala Leu Leu Ile MetGly Ser Phe Val Pro Trp Leu 145 150 155 160 Tyr Tyr Ser Phe Tyr Cys AsnPro Gln Pro Cys Phe Ile Tyr Leu Ile 165 170 175 Val Ile Cys Val Leu GlyIle Ala Ala Ile Ile Val Ser Gln Trp Asp 180 185 190 Met Phe Ala Thr ProGln Tyr Arg Gly Val Arg Ala Gly Val Phe Val 195 200 205 Gly Leu Gly LeuSer Gly Ile Ile Pro Thr Leu His Tyr Val Ile Ser 210 215 220 Glu Gly PheLeu Lys Ala Ala Thr Ile Gly Gln Ile Gly Trp Leu Met 225 230 235 240 LeuMet Ala Ser Leu Tyr Ile Thr Gly Ala Ala Leu Tyr Ala Ala Arg 245 250 255Ile Pro Glu Arg Phe Phe Pro Gly Lys Cys Asp Ile Trp Phe His Ser 260 265270 His Gln Leu Phe His Ile Phe Val Val Ala Gly Ala Phe Val His Phe 275280 285 His Gly Val Ser Asn Leu Gln Glu Phe Arg Phe Met Ile Gly Gly Gly290 295 300 Cys Thr Glu Glu Asp Ala Leu 305 310

1. A protein as set forth in (a) or (b) below: (a) a protein comprising an amino acid sequence according to Seq. No. 2, 4, 6 or 8, or (b) a protein comprising an amino acid sequence according to Seq. No. 2, 4, 6 or 8 with one or more amino acids deleted, replaced or added, and having adiponectin binding ability.
 2. A gene encoding the proteins according to claim
 1. 3. The gene according to claim 2, comprising DNA as set forth in (c) or (d) below: (c) DNA comprising a base sequence according to Seq. No. 1, 3, 5 or 7; (d) DNA which hybridizes under stringent conditions with DNA complementary to DNA comprising a base sequences according to Seq. No. 1, 3, 5 or 7, and which encodes a protein having adiponectin binding ability.
 4. A recombinant vector containing the gene according to claim 2 or
 3. 5. A transformant containing the recombinant vector according to claim
 4. 6. An antibody or fragment thereof capable of reacting with the protein according to claim
 1. 7. A screening method for a ligand, agonist or antagonist to an adiponectin receptor, comprising a step of bringing a test substance into contact with the protein according to claim
 1. 8. A screening kit for a ligand, agonist or antagonist to an adiponectin receptor, comprising the protein according to claim 1, the DNA according to claim 2 or 3, and the recombinant vector according to claim 4 or the transformant according to claim
 5. 